AU782230B2 - Methods of screening for compounds that modulate the LSR-leptin interaction and their use in the prevention and treatment of obesity-related diseases - Google Patents

Description

WO 01/21647 PCT/IB00/01470 1 METHODS OF SCREENING FOR COMPOUNDS THAT MODULATE THE LSR LEPTIN INTERACTION AND THEIR USE IN THE PREVENTION AND TREATMENT OF OBESITY-RELATED DISEASES FIELD OF THE INVENTION The present invention relates to the field of obesity research, in particular methods of screening for new compounds for the treatment of obesity and obesity-related diseases and disorders, as well as methods of treating obesity-related diseases and disorders. To this end, the characterization of the interaction between a new complex receptor polypeptide, LSR (Lipolysis Stimulated Receptor), and one of its ligands, leptin, is described. The obesity-related diseases or disorders envisaged to be treated by the methods of the invention include, but are not limited to, anorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension and syndrome X. In addition, and more generally, the various pathologies associated with abnormalities in the metabolism of cytokines, may be treated by the methods of the invention.

BACKGROUND OF THE INVENTION The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.

Obesity is a public health problem that is serious, widespread, and increasing. In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634). Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643). Even modest weight loss ameliorates these associated conditions.

While still acknowledging that lifestyle factors including environment, diet, age and exercise play a role in obesity, twin studies, analyses of familial aggregation, and adoption studies all indicate that obesity is largely the result of genetic factors (Barsh et al (2000) Nature 404:644- 651). In agreement with these studies, is the fact that an increasing number of obesity-related genes are being identified. Some of the more extensively studied genes include those encoding leptin (ob) and its receptor pro-opiomelanocortin (Pomc), melanocortin4-receptor (Mc4r), agouti protein carboxypeptidase E (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basic helix-loophelix 2 (Nhlh2), prohormone convertase 1 IPCSK1), and tubby protein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).

The gene encoding leptin, one of the most widely studied obesity genes, is involved in the mechanisms of satiety (rev'd in Schwartz et al (2000) Nature 404:661-671). Leptin is a plasma protein of 16 kDa produced by adipocytes (Zhang et al ((1994) Nature 372:425-432). Mice with an WO 01/21647 PCT/IB00/01470 2 autosomal recessive mutation in this gene (ob/ob.mice) are obese and hyperphagic. Similarly, mice with an autosomal recessive mutation of the leptin receptor (db/db mice, for example) are also obese (Campfield et al (1995) Science 269:546-549). Administration of leptin to ob/ob, but not db/db, mice corrects their relative hyperphagia and allows normalization of their weight (Weigle (1995) J. Clin. Invest. 96:2065-2070).

Leptin circulates in the body at levels proportional to body fat content (Considine et al (1996) New Eng J Med 334 :292-295) and enters the central nervous system (CNS) at levels proportional to the plasma level (Schwartz ct al (1996) Nature Med 2 :589-593). Leptin receptors are expressed by brain neurons involved in energy intake (Baskin et al (1999) Diabetes 48 :828- 833; Cheung et al (1997) Endocrinology 138:4489-4492) and administration of leptin into the brain reduces food intake (Weigle (1995) J. Clin. Invest. 96:2065-2070 Campfield et al (1995) Science 269:546-549), whereas its deficiency increases food intake (Zhang et al (1994) Nature 372:425- 432).

Despite this clear evidence ofleptin's role as an adiposity signal, with only a few exceptions the genes encoding leptin or its ob receptor have proved to be normal in obese human subjects (Kopelman et al (2000) Nature 404:635-643). Furthermore, and paradoxically, the plasma concentrations of leptin, are abnormally high in most obese human subjects (Considine et al (1996) New Eng J Med 334 :292-295).

SUMMARY OF THE INVENTION The present invention results from a focusing of the research effort on the discovery of the mechanisms of leptin elimination. The most widely accepted working hypothesis is that the plasma levels of leptin are high in obese subjects because this hormone is produced by adipose tissue which is increased in obese subjects. In contrast, although not wishing to be limited by any particular theory, the inventors postulated that the concentrations of leptin are increased in obese individuals because the clearance of this hormone is reduced. The resulting high levels of leptin cause a leptin resistance syndrome. Thus, the treatment of obese subjects should not be based on increasing leptin levels, but in normalizing leptin levels.

The lipolysis stimulated receptor (LSR) displays a high affinity for unmodified triglyceride-rich lipoproteins and is involved in the partitioning of dietary lipids among the liver, adipose tissue and muscle. The instant invention stems inter alia from studies of the role of LSR in modulating obesity. As part of the instant invention, leptin and the leptin fragment described herein were found to diminish the postprandial lipemic response in dbP'/dbP'" mice which lack the leptin OB receptor, thereby showing that leptin signaling can be independent of the OB receptor. Further, the instant invention stems from the discovery that leptin increases the activity of LSR, binds directly to LSR, and that leptin binding leads to leptin degradation. Although not WO 01/21647 PCT/IB00/01470 3 wishing to be bound by a particular theory, the link between leptin signaling and LSR suggests the post-prandial lipemic response in dbP"/db'" mice is modulated through this pathway.

In addition, the inventors have discovered that LSR is actually at least two receptors, one for triglyceride-rich lipoproteins, and one for leptin. The three subunits that make up LSR, a, p, and actually combine in at least two ways: a and P together make up the LSR receptor for triglyceride-rich lipoproteins, and a' is a necessary part of the LSR receptor for leptin, that may include 0 as well. Thus, it is now clear that assays can be designed for identifying modulators or receptors/binding partners/signalling cascade members that are specific for the triglyceride-related activity of LSR or for the leptin-related activity of LSR or both.

Further, the invention features the discovery of a 22 amino acid region of human leptin that modulates LSR activity in vitro and in vivo in the same way as the intact human leptin, thus allowing the use of only this critical region in assays for modulators of the leptin-LSR interaction, and new leptin receptors and binding partners. The new leptin fragment can also be used in disease treatment since it is active in mice at a physiologically-relevant level. In addition, the homologous region from mouse leptin was found to inhibit LSR activity in the human system, and is thus an LSR antagonist of the invention as well as being a powerful tool for identifying further modulators (both inhibitory and stimulatory) of LSR activity.

In a preferred aspect, the invention features a leptin polypeptide fragment that modulates the activity of LSR, comprising at least 4, but not more than 50 contiguous amino acids of any one of the leptin polypeptide sequences set forth in Figure 13, wherein said at least 4 and not more than 50 contiguous amaino acids comprise the leptin fragment central sequence. In preferred embodiments, the leptin polypeptide fragment comprises at least 10 but not more than at least 20 but not more than 40, or at least 20 but not more than 30 contiguous amino acids.

Alternatively, the invention features a variant of a leptin polypeptide fragment that modulates the activity of LSR, consisting of a 22 contiguous amino acid sequence that is at least identical to the leptin fragment variable region of any one of the leptin polypeptide sequences set forth in Figure 13. In preferred embodiments, the variant of a leptin polypeptide fragment is 85% identical, or 95% identical to the leptin fragment variable region. Preferably the leptin fragments and variants are from human or mouse leptin.

In a second aspect, the invention features, a chimeric oligonucleotide, comprising at least 9 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, wherein said at least 9 contiguous nucleotides comprise at least one amino acid codon selected from the group consisting of TTA, TTG, TCA, TCG, TAU, TAC, TGT, TGC, TGG, CAA, CAG, AGA, GAA, GAG, and GGA, and wherein a point WO 01/21647 PCT/IB00/01470 4 mutation is present in said codon such that said codon is a stop codon. Alternatively, the chimeric oligonucleotide comprises at least 9 contiguous nucleotides of SEQ ID NO:1, wherein said at least 9 contiguous nucleotides comprise a single nucleotide polymorphism selected from the group consisting of Al to A32.

In a third aspect, the invention features a zinc finger protein, comprising a DNA binding domain that binds specifically to 18 nucleotides of a sequence at least 50% homologous to SEQ ID NO:1, wherein said 18 nucleotides comprise two fragments of 9 contiguous nucleotides, and wherein said fragments are separated by 0, 1, 2, or 3 nucleotides. In preferred embodiments, said sequence is at least 50% homologous to intronic sequences selected from the group consisting of 2357 to 3539, 3885 to 12162, 12283 to 15143, 15201 to 17764, 15912 to 19578, 19753 to 19898, 19959 to 20055, 20188 to 20328, and 20958 to 21046 of SEQ ID NO:1, preferably to residues 2357 to 3539 of SEQ ID NO: 1, or alternatively 5' untranslated regions such as the sequence 1 to 2356 of SEQ ID NO:1. In preferred embodiments, said protein further comprises a functional domain selected from the group consisting of a transcription repressor and a transcription initiator; preferably said repressor is a KRAB repressor and said initiator is a VP16 initiator. In other preferred embodiments, said protein further comprises a small molecule regulatory system, preferably said system is selected from the group consisting of a Tet system, RU486, and ecdysone.

In a fourth aspect, the invention features polynucleotides encoding the leptin polypeptide fragments and variants of the invention, or polynucleotides encoding a zinc finger protein of the invention.

In a fifth aspect, the invention features recombinant vectors comprising the polynucleotides encoding the leptin polypeptide fragments and variants of the invention, or polynucleotides or recombionant vectors encoding a zinc finger protein of the invention. In preferred embodiments, said vector is an adenovirus associated virus.

In a sixth aspect, the invention features recombinant cells comprising the polynucleotides and recombinant vectors encoding the leptin polypeptide fragments and variants of the invention, or polynucleotides and recombinant vectors encoding zinc finger proteins of the invention. In preferred embodiments, the recombinant cell comprising the polynucleotides and recombinant vectors encoding leptin fragments and variants and zinc finger polypeptides of the invention, are transfected with at least one LSR polypeptide comprising a sequence at least identical to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. Preferably, said transfected cell is stably transfected. Preferably, said cell is selected from the group consisting of PLC, CHO-K 1, Hep3B, Hepa 1-6, and HepG2.

WO 01/21647 PCT/IB00/01470 In a seventh embodiment, the invention features a pharmaceutical composition comprising the leptin polypeptide fragments and variants of the invention, or polynucleotides or recombinant vectors encoding a zinc finger protein of the invention, or chimeraplasts of the invention.

In an eighth aspect, the invention features non-human mammals comprising polynucleotides and recombinant vectors encoding zinc finger proteins of the invention.

Preferably, said vector is an adenovirus associated virus.

In a ninth aspect, the invention features a method of treating or preventing an obesityrelated disease or disorder comprising providing to an individual in need of such treatment a pharmaceutical composition comprising the leptin polypeptide fragments and variants of the invention. Preferably, said disease is congenital generalized lipodystrophy. Alternatively, the patient is provided a chimeric oligonucleotide of the invention or a polynucleotide or recombinant vector encoding a zinc finger protein of the invention. Preferably, said providing comprises a liposome, and preferably said vector is an adenovirus associated virus. In preferred embodiments, the obesity related disease or disorder is selcted from the group consisting of obesity, anorexia, cachexia, cardiac insufficiency, coronary insufficiency, stroke, hypertenison, atheromatous disease, atherosclerosis, high blood pressure, non-insulin-dependent diabetes, hyperlipidemia, hyperuricemia, and Syndrome X. Preferably the individual is an animal, preferably a mammal, most preferably a human.

In a tenth aspect, the invention features a method of designing mimetics of a leptin fragment that modulates an activity of LSR, comprising: identifying critical interactions between one or more amino acids of said leptin fragment and LSR; designing potential mimetics to comprise said critical interactions; and testing said potential mimetics ability to modulate said activity as a means for designing said mimetics. Preferably, the leptin fragment consists of the leptin fragment variable region or the leptin fragment central sequence of any one of the leptin polypeptide sequences set forth in Figure 13. Alternatively, the leptin fragment is any one of the leptin fragments or variants of the invention. Preferably, the leptin fragment or variant is from human or mouse leptin. In preferred embodiments, the activity oflSR is selected from the group consisting of leptin binding, leptin uptake, leptin degradation, triglyceride binding, triglyceride uptake, and triglyceride degradation. Preferably the critical interactions are selected from the group consisting of hydrogen bonding, covalent bonding, Van der Waals forces, steric hindrances, and hydrophobic interactions, and are identified using assays selected from the group consisting of NMR, X-ray crystallography, and computer modeling.

In an eleventh aspect, the invention features a method of inhibiting the expression of at least one subunit of LSR, comprising providing to a cell a chimeric oligonucleotide of the invention that changes a amino acid codon to a stop codon. Preferably, the cell is selected from WO 01/21647 PCT/IB00/01470 6 the group consisting of PLC, CHO-K 1, HepG2, Hepa 1-6, and Hep3B. Alternatively the cell is in a mammal, preferably a mouse, more preferably in a human, and is provided using a liposome.

In a related aspect, the invention features a method of modulating the expression of at least one subunit of LSR, comprising providing to a cell a polynucleotide encoding a zinc finger protein of the invention. Preferably, said cell is selected from the group consisting of PLC, CHO-KI, HepG2, Hepa 1-6, and Hep3B. Alternatively, said cell is in an animal, preferably a mammal, and preferably said mammal is a mouse or a human.

In a twelfth aspect, the invention features a method for selecting a compound useful for the treatment or prevention of an obesity-related disease or disorder, comprising: contacting a recombinant cell comprising a polynucleotide or recombinant vector encoding a zinc finger protein of the invention, and that optionally further comprises at least one LSR polypeptide comprising a sequence at least 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, with a candidate compound; and detecting a result selected from the group consisting of a modulation of an activity of the Lipolysis Stimulated Receptor and modulation of expression of the Lipolysis Stimulated Receptor; as a means for selecting said compound useful for the treatment or prevention of said obesity-related disease or disorder. In preferred embodiments, said contacting is in the presence of a ligand of said Lipolysis Stimulated Receptor. Preferably, said ligand is selected from the group consisting of cytokine, lipoprotein, free fatty acids, Apml, and Clq. Most preferably said cytokine is leptin, or a leptin polypeptide fragment or variant of the invention. Alternatively said free fatty acid is oleate.

In preferred embodiments, said LSR activity is selected from the group consisting of binding of lipoproteins, uptake of lipoproteins, degradation of lipoproteins, binding of leptin, uptake of leptin, and degradation of leptin. Preferably said modulation is an increase in said activity, alternatively a decrease in activity. In other preferred embodiments, said expression is on the surface of said cell, and preferably said detecting comprises FACS. Preferably, said detecting further comprises antibodies that bind specifically to said LSR, wherein said LSR comprises an amino acid sequence at least 75% homologous to at least one of the sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19 Most preferably, said antibodies bind specifically to a region of said LSR selected from the group consisting of an amino terminus, a carboxy terminus, a splice site, a cytokine binding site, a fatty acid binding site, a clathrin binding site, an apoprotein ligand binding site, a LI/LL motif, a RSRS motif, and a hydrophobic region. Preferably, said cell is selected from the group consisting of PLC, CHO-KI, Hep3B, Hepa 1-6, and HepG2.

In other preferred embodiments, said candidate compound is selected from the group consisting ofpeptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, and small molecules. Preferably, said obesity-related diseases and disorders are selected from the group consisting of obesity, anorexia, cachexia, cardiac insufficiency, coronary insufficiency, stroke, hypertension, atheromatous disease, atherosclerosis, high blood pressure, non-insulin-dependent diabetes, hyperlipidemia, hyperuricemia, and Syndrome X.

In a thirteenth aspect, the invention features a method of selecting for genes that modulate an activity of the Lipolysis Stimulated Receptor, comprising: providing a retroviral gene library to cells that express said Lipolysis Stimulated Receptor; contacting said cells with a ligand of said Lipolysis Stimulated Receptor; detecting a change in said activity of the Lipolysis Stimulated Receptor as a means for selecting for said genes. In preferred embodiments, said retroviral gene library comprises a cDNA library from tissues selected from the group consisting of liver and adipose. Preferably, said retroviral gene library further comprises a detectable marker protein selected from ;the group consisting of GFP, truncated CD2, and truncated CD4. In other preferred embodiments, the invention further comprises selecting said cells comprising the retroviral gene library for moderate expression of GFP; preferably said selecting of cells is by FACS.

20 In other preferred embodiments, said ligand is selected from the group consisting of cytokine, lipoprotein, free fatty acids, Apml, and Clq. Most preferably •said cytokine is leptin, or a leptin polypeptide fragment or variant of the invention.

Alternatively said free fatty acid is oleate.

In yet other preferred embodiments, preferably said detecting a change in said 25 activity comprises FACS. Preferably, said detecting further comprises antibodies that bind specifically to said LSR, wherein said LSR comprises an amino acid sequence at least 75% homologous to at least one of the sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. Most preferably, said antibodies bind specifically to a region of said LSR selected from the group consisting of an amino terminus, a carboxy terminus, a splice site, a cytokine binding site, a fatty acid binding site, a clathrin binding site, an apoprotein ligand binding site, a LILL motif, a RSRS motif, and a hydrophobic region. Preferably, said cell is selected from the group consisting ofPLC, CHO-Kl, Hep3B, Hepa 1-6, and HcpG2.

In a fourteenth aspect, the present invention provides a leptin polypeptide fragment that modulates an activity of LSR, comprising at least 4, but not more than contiguous amino acids of a polypeptide sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, wherein said at least 4 and not more than contiguous amino acids include the leptin fragment central sequence.

In a fifteenth aspect, the present invention provides a polynucleotide encoding said leptin fragment of the fourteenth aspect, or the complement of a polynucleotide encoding said leptin fragment of the fourteenth aspect.

In a sixteenth aspect, the present invention provides a recombinant cell comprising said polynucleotide of the fifteenth aspect.

In a seventeenth aspect, the present invention provides a recombinant vector comprising said polynucleotide of the fifteenth aspect.

In an eighteenth aspect, the present invention provides a recombinant cell comprising said vector of the seventeenth aspect.

In a nineteenth aspect, the present invention provides a pharmaceutical composition comprising said leptin fragment of the fourteenth aspect and a pharmaceutically acceptable diluent.

20 In a twentieth aspect, the present invention provides a method of preventing or treating an obesity-related disease or disorder comprising providing to an individual in °need of such treatment said pharmaceutical composition of the nineteenth aspect.

In a twenty-first aspect, the present invention provides a method of designing mimetics of a leptin fragment that modulates an activity of LSR, comprising: 1* 25 identifying critical interactions between one or more amino acids of said S(*b leptin fragment of the fourteenth aspect and LSR; designing potential mimetics to comprise said critical interactions; and testing said potential mimetics ability to modulate said activity as a means for designing said mimetics.

In a twenty-second aspect, the present invention provides for the use of a leptin polypeptide fragment of the fourteenth aspect for the manufacture of a medicament for preventing or treating an obesity-related disease or disorder in an individual.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

DETAILED DESCRIPTION OF THE INVENTION LSR (Lipolysis Stimulated Receptor), which is described in PCT publication No WO IB98/01257 (hereby incorporated by reference herein in its entirety including any figures, tables, *oo **oo *o oo WO 01/21647 PCT/IB00/01470 8 or drawings), is expressed on the surface of hepatic cells, and is involved in the partitioning of dietary lipids between the liver and peripheral tissues, including muscles and adipose tissue. The LSR gene encodes, by alternative splicing, three types of subunits, LSR a, LSR and LSR 3.

The a' subunit specifically binds a cytokine, leptin, which activates LSR and is taken up and degraded. The invention is drawn inter alia to compounds that modulate the interaction between LSR and leptin useful in the treatment or prevention of obesity-related diseases and disorders.

Definitions Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.

As used interchangeably herein, the terms "oligonucleotides", and "polynucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The terms "nucleotide", "nucleotide sequence" and "nucleic acid" are used herein consistently with their use in the art, including to encompass "modified nucleotides" which comprise at least one modification, including by way of example and not limitation: an alternative linking group, an analogous form of purine, an analogous form of pyrimidine, or an analogous sugar. For examples of analogous linking groups, purines, pyrimidines, and sugars see for example PCT publication No. WO 95/04064. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.

The terms polynucleotide construct, recombinant polynucleotide and recombinant polypeptide are used herein consistently with their use in the art. The terms "upstream" and "downstream" are also used herein consistently with their use in the art. The terms "base paired" and "Watson Crick base paired" are used interchangeably herein and consistently with their use in the art. Similarly, the terms "complementary", "complement thereof", "complement", "complementary polynucleotide", "complementary nucleic acid" and "complementary nucleotide sequence" are used interchangeably herein and consistently with their use in the art.

The term "purified" is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide). Purified can also refer to the separation of covalently closed polynucleotides from linear polynucleotides, or vice versa, for example. A polynucleotide is substantially pure when at least about 50%, 60%, 75%, or 90% of a sample contains a single WO 01/21647 PCT/IB00/01470 9 polynucleotide sequence. In some cases this involves a determination between conformations (linear versus covalently closed). A substantially pure polynucleotide typically comprises about 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample. Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

Similarly, the term "purified" is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins. In some preferred embodiments, a polypeptide is substantially pure when at least about 50%, 60%, 75%, 85%, 90%, or 95% of a sample exhibits a single polypeptide sequence. In some preferred embodiments, a substantially pure polypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, or 99% weight/weight of a protein sample. Polypeptide purity or homogeneity is indicated by a number of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other methods well known in the art.

Further, as used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Alternatively, purification may be expressed as "at least" a percent purity relative to heterologous polynucleotides (DNA, RNA or both) or polypeptides. As a preferred embodiment, the polynucleotides or polypeptides of the present invention are at least; 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure relative to heterologous polynucleotides or polypeptides. As a further preferred embodiment the polynucleotides or polypeptides have an "at least" purity ranging from any number, to the thousandth position, between 90% and 100% at least 99.995% pure) relative to heterologous polynucleotides or polypeptides. Additionally, purity of the polynucleotides or polypeptides may be expressed as a percentage (as described above) relative to all materials and compounds other than the carrier solution. Each number, to the thousandth position, may be claimed as individual species of purity.

The term "isolated" requires that the material be removed from its original environment the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such WO 01/21647 PCT/IB00/01470 polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.

SSpecifically excluded from the definition of "isolated" are: naturally occurring chromosomes chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist cither as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein a 5' EST makes up less than 5% of the number of nucleic acid inserts in the vector molecules. Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymatically digested). Further specifically excluded are the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).

The term "primer" denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.

The term "probe" denotes a defined nucleic acid segment (or nucleotide analog segment, PNA as defined hereinbelow) which can be used to identify a specific polynucleotide sequence present in a sample, said nucleic acid segment comprising a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.

The term "polypeptide" refers to a polymer of amino acids without regard to the length of the polymer. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides. For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition arc polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

Without being limited by theory, the compounds/polypeptides of the invention are believed to treat "diseases involving the partitioning of dietary lipids between the liver and WO 01/21647 PCT/IB00/01470 11 peripheral tissues The term "peripheral tissues" is meant to include muscle and adipose tissue.

In preferred embodiments, the compounds/polypeptides of the invention partition the dietary lipids toward the muscle. In alternative preferred embodiments, the dietary lipids are partitioned toward the adipose tissue. In other preferred embodiments, the dietary lipids are partitioned toward the liver. In yet other preferred embodiments, the compounds/polypeptides of the invention increase or decrease the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle. Dietary lipids include, but are not limited to triglycerides and free fatty acids.

Preferred diseases believed to involve the partitioning of dietary lipids include obesity and obesity-related diseases and disorders such as atherosclerosis, heart disease, insulin resistance, hypertension, stroke, Syndrome X, and Type I diabetes. Type II diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDSrelated weight loss, neoplasia-related weight loss, anorexia, and bulimia.

The term "obesity" as used herein is defined in the WHO classifications of weight (Kopelman (2000) Nature 404:635-643). Underweight is less than 18.5 (thin); Healthy is 18.5- 24.9 (normal); grade 1 overweight is 25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade 3 overweight is greater than or equal to 40.0 BMI (morbid obesity). BMI is body mass index and is kg/m 2 Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk. Similarly for women, greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk. The waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis. Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers, and bioimpedance, which is based on the principle that lean mass conducts current better than fat mass because it is primarily an electrolyte solution; measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.

The term "agent acting on the partitioning of dietary lipids between the liver and peripheral tissues" refers to a compound or polypeptide of the invention that modulates the partitioning of dietary lipids between the liver and the peripheral tissues as previously described.

Preferably, the agent increases or decreases the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle. Preferably the agent decreases or increases the body weight of WO 01/21647 PCT/IB00/01470 12 individuals or is used to treat or prevent an obesity-related disease or disorder such as atherosclerosis, heart disease, insulin resistance, hypertension, stroke, Syndrome X, and Type II diabetes. Type H diabetes-related complications to be treated by the methods of the invention include, but are not limited to, microangiopathic lesions, ocular lesions, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, anorexia, and bulimia.

The terms "response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues refer to drug efficacy, including but not limited to, ability to metabolize a compound, to the ability to convert a pro-drug to an active drug, and to the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptorrelated) of a drug in an individual.

The terms "side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. "Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.

As used herein, the term "antibody" refers to a polypeptide or group ofpolypeptides which are comprised of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, and that allows an immunological reaction with the antigen. Antibodies include recombinant proteins comprising the antibody binding domains, as well as fragments, including Fab, Fab', F(ab)2, and F(ab')2 fragments.

As used herein, an "antigenic determinant" is the portion of an antigen molecule, in this case an LSR polypeptide, that determines the specificity of the antigen-antibody reaction. An "epitope" refers to an antigenic determinant of a polypeptide. An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids.

Methods for determining the amino acids which make up an epitope include x-ray WO 01/21647 PCT/IB00/01470 13 crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by H. Mario Geysen et al. 1984. Proc. Natl. Acad. Sci. U.S.A.

81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506.

The term "compound" as used herein refers to molecules, either organic or inorganic, that can be tested for activity in an assay. Preferably, compounds have a low molecular weight of less than 500 kda, some compounds can have a molecular weight between 500 and 1500, other compounds may have a molecular weight of at least 1500 kda. In addition, compounds of interest preferably have a desired activity at a low concentration, e.g. a compound that is active at a concentration of 1 ng/mL or less, is generally preferred over one that is active at 1 ng/mL to 100 ng/mL, or one that is active only at concentrations greater than 100 ng/mL. Examples of compounds to be tested in the assays herein include: peptides, peptide libraries, non-peptide libraries, antibodies, and peptoids.

The term "activity" as used herein refers to a measurable result of the interaction of molecules. For example, some LSR activities include leptin binding, leptin uptake, leptin degradation, as well as triglyceride binding, triglyceride uptake, and triglyceride degradation.

Some exemplary methods of measuring these activities are provided herein.

The term "modulate" as used herein refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds in the assays, activities can increase there could be increased levels of leptin binding), or "decrease" there could be decreased levels of leptin binding) as compared to controls in the absence of these compounds. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. Similarly, a decrease in activity is preferably at least more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. A compound that increases a known activity is an "agonist". One that decreases, or prevents, a known activity is an "antagonist".

The term "monitoring" as used herein refers to any method in the art by which an activity can be measured. For each of the activities in the assays of the invention, exemplary methods are provided in the Examples section.

The term "providing" as used herein refers to any means of adding a compound or molecule to something known in the art. Examples of providing can include the use of pipets, pipettmen, syringes, needles, tubing, guns, etc. This can be manual or automated. It can include transfection by any mean or any other means of providing nucleic acids to dishes, cells, tissue, cell-free systems and can be in vitro or in vivo. Methods are provided in the Examples section as examples.

WO 01/21647 PCT/IB00/01470 14 The term "LSR-related diseases and disorders" as used herein refers to any disease or disorder or condition comprising an aberrant functioning of LSR, or a subunit(s) of LSR, to include aberrant levels of expression of LSR, or a subunit(s) of LSR (either increased or decreased), aberrant activity of LSR (either increased or decreased), and aberrant interactions with ligands or binding partners (either increased or decreased). By "aberrant" is meant a change from the type, or level of activity seen in normal cells, tissues, or individuals, or seen previously in the cell, tissue, or individual prior to the onset of the illness.

The term "cosmetic treatments" is meant to include treatments with compounds or polypeptides of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically thin. Thus, these individuals have a body mass index (BMI) below the cut-off for clinical obesity below 25 kg/mZ) and above the cut-off for clinical thinness above 18.5 kg/m2). In addition, these individuals are preferably healthy do not have an obesity-related disease or disorder of the invention). "Cosmetic treatments" are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example. These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.

The term "preventing" as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with obesity or LSR.

The term "treating" as used herein refers to administering a compound after the onset of clinical symptoms.

The term "in need of treatment" as used herein refers to ajudgment made by a caregiver physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.

The term "perceives a need for treatment" refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under "cosmetic treatment" above. The term "perceives a need for treatment" in other embodiments can refer to the decision that an owner of an animal makes for cosmetic treatment of the animal.

The term "individual" as used herein refers to a mammal, including animals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, most preferably humans.

WO 01/21647 PCT/IB00/01470 The term "non-human animal" refers to any non-human vertebrate, birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice. Both the terms "animal" and "mammal" expressly embrace human subjects unless preceded with the term "non-human".

The terms "percentage of sequence identity" and "percentage homology" are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad.

Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol.. 215(3):403-410) Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al., 1996, Methods Enzymol. 266:383- 402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267- 2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402. In particular, five specific BLAST programs are used to perform the following task: BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; BLASTN compares a nucleotide query sequence against a nucleotide sequence database; BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

WO 01/21647 PCT/IB00/01470 16 The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61. Less preferably, the PAM or PAM250 matrices may also be used (see, Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).

By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 0 C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 gg/mL denatured salmon sperm DNA. Filters are hybridized for 48 h at 65 the preferred hybridization temperature, in prehybridization mixture containing 100 pg/mL denatured salmon sperm DNA and 5-20 X 106 cpm of "P-labeled probe. Alternatively, the hybridization step can be performed at 65 OC in the presence of SSC buffer, 1 x SSC corresponding to 0.15 M NaCI and 0.05 M Na citrate.

Subsequently, filter washes can be done at 37 OC for I h in a solution containing 2 x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1 X SSC at 50 °C for min. Alternatively, filter washes can be performed in a solution containing 2 x SSC and 0.1% SDS, or 0.5 x SSC and 0.1% SDS, or 0.1 x SSC and 0.1% SDS at 68 oC for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography.

Other conditions of high stringency that may be used are well known in the art (see, for example, Sambrook et al., 1989; and Ausubel et al., 1989). These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. A person of ordinary skill in the art will realize that the hybridization conditions described above are to be adapted according to the length of the desired nucleic acid following techniques well known to the one skilled in the art. Suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (1985) or in Sambrook et al.(1989).

Variants WO 01/21647 PCT/IB00/01470 17 It will be recognized by one of ordinary skill in the art that some amino acids of the polypeptide sequences of the present invention can be varied without significant effect on the structure or function of the protein; there will be critical amino acids in the polypeptide sequence that determine activity. Thus, the invention further includes variants ofpolypeptides. Such variants include polypeptide sequences with one or more amino acid deletions, insertions, inversions, repeats, and substitutions either from natural mutations or human manipulation selected according to general rules known in the art so as to have little effect on activity.

Guidance concerning how to make phenotypically silent amino acid substitutions is provided below.

There are two main approaches for studying the tolerance of an amino acid sequence to change (See, Bowie, J. U. et al. 1990). The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions ofa cloned gene and selections or screens to identify sequences that maintain functionality.

These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions and indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described by Bowie et al. (supra) and the references cited therein.

Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Phe; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe, Tyr. In addition, the following groups of amino acids generally represent equivalent changes: Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; Cys, Ser, Tyr, Thr; (3) Val, lie, Leu, Met, Ala, Phe; Lys, Arg, His; Phe, Tyr, Trp, His.

Similarly, amino acids in polypeptide sequences of the invention that are essential for function can also be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (See, Cunningham et al. 1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for obesity-related activity using assays as described above. Of special interest are substitutions of charged amino acids with other charged or neutral amino acids that may produce proteins with highly desirable improved characteristics, such as less aggregation.

Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical WO 01/21647 PCT/IB00/01470 18 formulations, because aggregates can be immunogenic, (See, Pinckard, et al., 1967; Robbins, et al., 1987;and Cleland, et al., 1993).

Thus, the fragment, derivative, analog, or homolog of the polypeptide of the present invention may be, for example: one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code: or (ii) one in which one or more of the amino acid residues includes a substituent group: or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol): or (iv) one in which the additional amino acids are fused to the above form of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the polypeptide or a pro-protein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and not more than 20 conservative amino acid substitutions. Also provided are polypeptides which comprise the amino acid sequence of polypeptide, having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or I conservative amino acid substitutions.

Another specific embodiment of a modified polypeptide of the invention is a polypeptide that is resistant to proteolysis, for example a polypeptide in which a -CONH- peptide bond is modified and replaced by one or more of the following: a (CH2NH) reduced bond; a (NHCO) retro inverso bond; a (CH2-O) methylene-oxy bond; a (CH2-S) thiomethylene bond; a (CH2CH2) carba bond; a (CO-CH2) cetomethylene bond; a (CHOH-CH2) hydroxyethylene bond); a (N-N) bound; a E-alcene bond; or a -CH=CH- bond. Thus, the invention also encompasses a polypeptide or a fragment or a variant thereof in which at least one peptide bond has been modified as described above.

In addition, amino acids have chirality within the body of either L or D. In some embodiments it is preferable to alter the chirality of the amino acids in the polypeptides of the invention in order to extend half-life within the body. Thus, in some embodiments, one or more of the amino acids are preferably in the L configuration. In other embodiments, one or more of the amino aicds are preferably in the D configuration.

WO 01/21647 PCT/IB00/01470 19 I .Leptin Polynucleotides of the Invention Polynucleotides have been designed that encode a LSR-binding/activating/modulating portion of the leptin protein. This region was identified by a comparison of the human and murine amino acid sequences, and its activity was confirmed in vitro and in vivo (See Examples The recombinant polynucleotide encoding the LSR-activating leptin fragment can be used in a variety of ways, including: to express the polypeptide in recombinant cells so as to be purified and used as described below, to express the polypeptide in cells as part of an assay system to discover modulators of the leptin/LSR interaction, and as part of a gene surgery where the fragment itself can be used in treatment and/or prevention of obesityrelated diseases and disorders and modulating body mass.

The invention relates to the polynucleotides encoding a leptin polypeptide fragment described in the Examples (7 and variants and fragments thereof as described herein in Leptin Polypeptides of the Invention (section II), as well as to variants and fragments of the polynucleotides that encode these polypeptides. Preferably, polynucleotides are purified, isolated and/or recombinant.

In other preferred embodiments, variants of the leptin polynucleotides encoding leptin polypeptides as described herein in Leptin Polypeptides of the Invention are envisioned.

Variants of polynucleotides, as the term is used herein, are polynucleotides whose sequence differs from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.

Variants of leptin polynucleotides according to the invention may include, without being limited to, nucleotide sequences which are at least 90% (preferably at least 95%, more preferably at least 99%, and most preferably at least 99.5%) identical to a polynucleotide that encodes a leptin polypeptide of the invention, or to any polynucleotide fragment of at least 8 (preferably at least 15, more preferably at least 25, and most preferably at least 45) consecutive nucleotides of a polynucleotide that encodes a polypeptide of the invention.

Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. Alterations in the leptin coding regions of the invention may produce conservative or non-conservative amino acid substitutions, deletions or WO 01/21647 PCT/IB00/01470 additions in the encoded protein. Preferably, the nucleotide substitutions result in nonconservative amino acid changes and more preferably in conservative amino acid changes in the encoded polypeptide.

In cases where the nucleotide substitutions result in one or more amino acid changes, preferred leptin polypeptides include those that retain the same activities and activity levels as the leptin polypeptide encoded by the reference polynucleotide sequence, as well as those where the level of one or more activities is increased, and alternatively where the level of one or more activities is decreased or even absent. Leptin polypeptide activities of the invention are described herein in the Examples in more detail 10 14), but include LSR binding leading to the uptake and degradation of leptin, as well as the upregulation of LSR receptors that bind, uptake and degrade triglycerides. Examples of assays to determine the presence or absence of specific leptin activities and the level of the activity(s) are also described herein.

By "retain the same activities" is meant that the activity measured using the polypeptide encoded by the variant leptin polynucleotide in assays is at least 75% (preferably at least more preferably at least 95%, most preferably at least 98%) and not more than 125% (preferably not more than 115%, more preferably not more than 105%, most preferably not more than 102%) of the activity measured using the leptin polypeptide encoded by the reference sequence.

By the activity being "increased" is meant that the activity measured using the polypeptide encoded by the variant leptin polynucleotide in assays is at least 125% (preferably at least 150%, more preferably at least 200%, most preferably at least 500%) of the activity measured using the leptin polypeptide encoded by the reference sequence.

By the activity being "decreased" is meant that the activity measured using the polypeptide encoded by the variant leptin polynucleotide in assays is not more than (preferably not more than 50%, more preferably not more than 25%, most preferably not more than 10%) of the activity measured using the leptin polypeptide encoded by the reference sequence.

By the activity being "absent" is meant that the activity measured using the polypeptide encoded by the variant leptin polynucleotide in assays is less than 25%, alternatively less than (preferably less than more preferably less than most preferably less than of the activity measured using the leptin polypeptide encoded by the reference sequence.

A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part, but not all, of a given nucleotide sequence, preferably the nucleotide sequence encoding a leptin polypeptide that binds and activates LSR, and variants thereof as described above, and the complements of these polynucleotides. Such fragments may be "free-standing", i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger WO 01/21647 PCT/IB00/01470 21 non-leptin polynucleotide of which they form a part or region. However, several fragments may be comprised within a single larger polynucleotide.

Optionally, such fragments may consist of a contiguous span that ranges in length from 8, 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80 ,90, 10, 110, 120, 130, 140, or 150 nucleotides in length.

A preferred embodiment of the invention includes isolated, purified, or recombinant polynucleotides consisting of a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides encoding a leptin polypeptide of the invention, or the complements thereof, wherein said contiguous span encodes a fragment of leptin that retains the same activities and activity levels as the leptin polypeptide encoded by the reference polynucleotide sequence, or encodes a fragment of leptin where the level of one or more activities is increased, or alternatively where the level of one or more activities is decreased or even absent as described above.

An additional preferred embodiment of the invention includes isolated, purified, or recombinant polynucleotides consisting of a contiguous span of 8 to 50 nucleotides of a leptin polypeptide of the invention, or their variants, or the complements thereof, wherein said contiguous span encodes a fragment of leptin that retains the same activities and activity levels as the leptin polypeptide encoded by the reference polynucleotide sequence, or encodes a fragment of leptin where the level of one or more activities is increased, or alternatively where the level of one or more activities is decreased or even absent as described above. Any of the above-described fragments may be comprised within a larger non-leptin polynucleotide fragment.

I. Leptin Polypeptide Fragments of the Invention Leptin polypeptide fragments that bind/activate/modulate LSR have been identified (Examples This region was identified by a comparison of the human and murine leptin amino acid sequences, and its activity confirmed in vitro and in vivo (See Examples The advantages to having identified a leptin fragment responsible for leptin activity, include its use as part of an assay system to discover leptin receptors and binding partners (in association with LSR for example), as a lead molecule for the design of other compounds able to modulate LSR activity, and as part of a treatment and/or prevention for obesity-related diseases and disorders. Knowledge of specific polypeptides involved is especially useful since it allows its use in assay systems (rather than the entire protein) and keeps the cost down (easily synthesized). In addition, a peptide can be expected to easily crystallize in the correct conformation to allow structure-function studies to design other small molecule activators.

WO 01/21647 PCT/IB00/01470 22 Finally, use of just the active portion in treatment should increase the chances of the peptide remaining active and potentially decreasing side-effects.

Furthermore, in the process of identifying the "active" portion of human leptin for human cells, a corresponding inhibitory portion of mouse leptin for human cells was identified.

Comparisons between the two highly similar fragments will enable the identification of important residues for both increasing the activity of LSR and inhibiting the activity of LSR.

This will be useful both in competitive assays for inhibitors and activators of LSR, and also for treatments in mammals and animals where inhibition of LSR is desired.

The invention relates to leptin polypeptides as well as to variants, fragments, analogs and derivatives of the leptin polypeptides described herein, including modified leptin polypeptides.

Preferred embodiments of the invention feature a leptin polypeptide that consists of a sequence described in Example 10, or variants, fragments, analogs, or derivatives thereof. Preferably the polypeptides are, purified, isolated and/or recombinant.

In other preferred embodiments, the invention features a leptin polypeptide fragment that modulates the activity of LSR, comprising at least 4, but not more than 50 contiguous amino acids of any one of the leptin polypeptide sequences set forth in Figure 13, wherein said at least 4 and not more than 50 contiguous amino acids comprise the leptin fragment central sequence.

By the leptin fragment central sequence as used herein is meant the four variable amino acids of the active leptin peptide identified in Example 10 by sequence comparisons and molecular modeling. These residues comprise ETLD (SEQ ID NO:40) and QKPE (SEQ ID NO:41) for the human and mouse sequences, respectively, in Fig. 13. Preferably, the leptin polypeptide fragment comprises at least 10, but not more than 50, more preferably at least 15 but not more than 40, or at least 20 and not more than 40, or most prefereably at least at least 15 but not more than 30, or 20 but not more than 30 contiguous amino acids of any one of the leptin polypeptide sequences set forth in Figure 13, wherein said contiguous amino acids comprise the leptin polypeptide variable region. Preferably the leptin polypeptide fragment is human or mouse, but most preferably human, or a derivative or variant thereof.

Variant leptin polypeptides of the invention may be 1) ones in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) ones in which one or more of the amino acid residues includes a substituent group, or 3) ones in which a modified leptin polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) ones in which the additional amino acids are fused to a modified leptin polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the modified leptin WO 01/21647 PCT/IB00/01470 23 polypeptide or a pre-protein sequence. Such variants are deemed to be within the scope of those skilled in the art.

Amino acid changes present in a variant polypeptide may be non-conservative amino acid changes but more preferably are conservative amino acid changes. In cases where there are one or more amino acid changes, preferred leptin polypeptides include those that retain the same activities and activity levels as the reference leptin polypeptide sequence, as well as those where the level of one or more activities is increased, and alternatively where the level of one or more activities is decreased or even absent. Assays for determining leptin polypeptide activities of the invention are described herein in the Examples (1-8 13) in more detail, but include LSR binding leading to the uptake and degradation of leptin, as well as the upregulation of LSR receptors that bind, uptake and degrade triglyceride-rich lipoproteins. Examples of assays to determine the presence or absence of specific leptin activities and the level of the activity(s) are also described herein. Dcfnitions of activities are provided in "Leptin Polynucleotides of the Invention" (section I).

In preferred embodiments, the invention features a variant of a leptin polypeptide fragment that modulates the activity of LSR, consisting of a 22 contiguous amino acid sequence that is at least 75% identical to the leptin fragment variable region of any one of the leptin polypeptide sequences set forth in Figure 13. By the leptin fragment variable region as used herein is meant the region of 22 amino acids that is shaded in figure 13 for all the species in the alignment. Preferably, the 22 contiguous amino acid sequence is at least 85% identical to the leptin fragment variable region of any one of the leptin polypeptide sequences set forth in Figure 13, more preferably 90% identical, most preferably 95% identical and optionally 100% identical.

Preferably the sequence is human or mouse, and most preferably human.

In yet other preferred embodiments, the invention features a variant of a leptin polypeptide fragment that modulates the activity of LSR, consisting of a 22 contiguous amino acid sequence, wherein at least 16 of the 22 amino acids are identical to the leptin fragment variable region of any one of the leptin polypeptide sequences set forth in Figure 13. Preferably, at least 18 of the 22 amino acids are identical to the leptin fragment variable region of any one of the Icptin polypeptide sequences set forth in Figure 13, more preferably 20 of the 22 are identical, most preferably all of the amino acids are identical. Preferably the sequence is human or mouse, and most preferably human.

A polypeptide fragment is a polypeptide having a sequence that is entirely the same as part, but not all, of a given polypeptide sequence, preferably a polypeptide encoded by a leptin gene and variants thereof. Such fragments may be "free-standing", i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger non-leptin polypeptide of which they form a part or region. However, several fragments may be comprised within a single WO 01/21647 PCT/IB00/01470 24 larger polypeptide. As representative examples ofpolypeptide fragments of the invention, there may be mentioned those which have from about 4, 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or to 55 amino acids long. Preferred are those fragments containing at least one amino acid substitution or deletion in a leptin polypeptide.

The present invention is particularly focused on a set of variant leptin polypeptides and the fragments thereof. A preferred set of polypeptides of the invention include isolated, purified, or recombinant polypeptides comprising a contiguous span of at least 3 (preferably at least 6, more preferably at least 10, most preferably at least 15) amino acids of any of the leptin fragment variable regions of the sequences provided in Figure 13.

I. Zinc Finger Proteins of the Invention Zinc finger proteins of the Cys2His2 type are malleable DNA binding proteins that can be designed to bind diverse sequences, and that typically contain 3 zinc finger domains. The inventors contemplate the use of any zinc finger protein engineered to bind the DNA of interest, specifically. Although six-fingered proteins have been described to target unique sites within the genome (International Publication WO 98/54311) proteins with different numbers of fingers that are engineered to bind specifically to the genome are also included in the invention. The six-fingered proteins described in WO 98/54311, bind two 9 contiguous base pair fragments (separated by 0, 1, 2, or 3 nucleotides) of DNA or RNA in a sequence specific fashion, and can be used to regulate gene transcription. The zinc finger proteins of the invention also include those that are designed to bind sequences a greater distance apart and thereby confer greater specificity with fewer (or the same number, or more) "fingers". Methods for designing the zinc finger proteins of the invention, as well as for determining the sequences to which the zinc finger proteins bind, are described in International Publication WO 98/54311 entitled "Zinc Finger Protein Derivatives and Methods Therefor".

For one embodiment of the invention, zinc finger proteins have been designed that will bind to the 5' regulatory regions and selected introns of LSR and thereby inhibit or augment the transcription of endogenous LSR as described herein (Example 12). Exogenous LSR that is introduced into the cell without these regulatory regions or introns (cDNA) will be expressed normally. This can be useful in vitro both as a research tool to study the role of the various LSR components in leptin signaling and triglyceride-rich lipoprotein uptake and degradation, for example, and as part of an assay to discover modulators of LSRlep and LSRtg activity.

Therefore, in currently preferred embodiments, zinc finger proteins are not designed to bind to the exons of LSR. However, in circumstances where no endogenous nor exogenouslyintroduced LSR activity is desired in a cell, for example, zinc finger proteins designed to bind to LSR exons could be useful.

WO 01/21647 PCT/IB00/01470 The invention features a zinc finger protein, comprising a DNA binding domain that binds specifically to 18 nucleotides of a sequence at least 50% homologous to SEQ ID NO:1, wherein said 18 nucleotides comprise two fragments of 9 contiguous nucleotides, and wherein said fragments are separated by 0, 1, 2, or 3 nucleotides. In preferred embodiments, the zinc finger protein binds to sequences that are at least 50% homologous to the sequence of the introns of SEQ ID NO:1. Preferably, the sequence is at least 50% homologous to the sequence of the first intron of SEQ ID NO: 1. In other preferred embodiments, the zinc finger protein binds specifically to 18 nucleotides of a sequence that is identical, 80%, 85%, or 90% identical, or most preferably 99 to 100% identical to SEQ ID NO :1, the introns of SEQ ID NO or preferably the first intron of SEQ ID NO :1.

In preferred embodiments of the invention, the zinc finger protein of the invention further comprises a functional domain selected from the group consisting of a transcription repressor and a transcription initiator. These repressors and initiators can be any that are known in the art. Preferably, the repressor is a KRAB repressor and the initiator is a VP16 initiator. In highly preferred embodiments, the protein further comprises a small molecule regulatory system that can be any known in the art; however, the system is preferably selected from the group consisting of a Tet system, RU486, and ecdysone.

It is envisioned that zinc finger proteins could be designed to bind to any 18 or more contiguous base pairs of a sequence at least 50%, preferably 75%, more preferably 90%, most preferably identical to the 5' regulatory region (for example, residues 1-2000 of SEQ ID NO: I) or any of the introns of LSR (for example, 2357 to 3539, 3885 to 12162, 12283 to 15143, 15201 to 17764, 15912 to 19578, 19753 to 19898, 19959 to 20055, 20188 to 20328, and 20958 to 21046 of SEQ ID NO:1), and more preferably residues 2357 to 3539 of SEQ ID NO:1. In particular, introns within 3,000 base pairs of the LSR start site are preferred, for example introns 1 through 3.

Guidance is available for determining optimal base pair stretches for zinc finger protein binding, and for determining what zinc finger amino acids will bind to what DNA sequences (WO 98/54311). This information has been used to design an algorithm for designing zinc finger proteins available from Sangamo BioSciences. However, as described in WO 98/54311, zinc finger proteins for binding a given piece of DNA can be identified by screening or "panning" libraries of zinc finger proteins with the DNA sequence. Zinc finger libraries can be made, for example, by randomly mutating genes encoding known zinc finger proteins (WO 98/54311). The effectiveness of the zinc finger protein identified by the panning procedure can then be assessed in the E. coli method described in WO 98/54311 (co-transfection of genes encoding the zinc finger protein and the gene of which the DNA sequence makes up a part).

The effectiveness of the zinc finger protein for inhibiting LSR expression can be further tested using the assay systems described in the Examples in particular the use of FACS following staining with an LSR specific antibody and quantitative PCR will be useful.

WO 01/21647 PCT/IBOO/01470 26 In preferred embodiments, addition of the zinc finger protein preferably inhibits LSR transcription completely, or inhibits LSR translation completely. By "inhibits transcription completely" is meant that the level of transcription following addition of the zinc finger protein is preferably below the level of detection by the assay used as compared to control cells. The assay used may be a Northern blot, or any other assay that measures RNA expression, such as quantitative PCR. Alternatively, the level of transcription of LSR may be significantly reduced.

By "significantly reduced" is meant that the amount of RNA is preferably reduced at least 2fold, more preferably at least 5-fold, and most preferably at least 10-fold compared to the level RNA prior to the addition of the zinc finger protein, or the level in control cells.

Similarly, by "inhibits translation completely" is meant that LSR protein is preferably below the level of detection by the assay used compared with control cells. The assay used may be a Western blot, or dot blot, or other type of immunoassay for example, or any other assay known in the art to be used to measure or detect the presence of proteins, such as FACS with fluorescent antibodies to LSR. Alternatively, the level of translation of LSR may be significantly reduced. By "significantly reduced" is meant the amount of protein present is preferably reduced at least 2-fold, more preferably at least 5-fold, most preferably at least fold compared to the level of protein present prior to the addition of zinc finger protein, or in control cells.

Highly preferred sequences to be used for designing zinc finger proteins include, residues 1841 to 1860, 1880 to 1898, 1918 to 1945, 1951 to 1973, and 3362 to 3382 of human LSR (SEQ ID NO: 1) and of the homologous regions in genes coding for LSR proteins of other species, preferably including mouse and rat LSR. The genomic sequences encoding LSR from other species can be identified by methods well-known in the art.

These zinc finger proteins can also be useful in vivo both as part of an assay system in animal models to discover modulators of LSRlep (at least may include P and/or a) and LSRtg (at least a, may include P and/or activity, as well as in gene surgery in which transcription of endogenous LSR is inhibited as part of the treatment for an obesity-related disease or disorder. This could be useful in a case where the LSR message was being overexpressed, or incorrectly expressed (mutated), for example. A potential therapy would include providing this zinc finger protein alone, in cases of simple over-expression, or in conjunction with other appropriate components of LSR if the cellular LSR was mutated. These proteins could be targeted to the appropriate cells (those with LSR) by using liposomes, for example, with leptin or another LSR binding protein in the liposome membrane.

In an alternative embodiment of the invention, zinc finger proteins are designed to bind to the 5' regulatory regions of LSR and thereby increase the transcription of endogenous LSR.

Typically, within the 5' regulatory region of genes are promoters as well as other regulatory WO 01/21647 PCT/IB00/01470 27 elements. Binding of zinc finger proteins to certain regions of the DNA may serve to facilitate binding of the initiation complex and thus transcription of the gene. For instance, where some unusual folding prevents access to the promoter region, if a zinc finger protein were to bind the DNA upstream such that the folding were prevented, then the promoter would have greater access and enhanced transcription should result. Alternatively, it may be possible to design a zinc finger protein that binds the promoter region directly, thereby initiating transcription.

In these and other circumstances, zinc finger binding proteins designed to bind stretches of DNA in the 5'regulatory region as described above can be tested for their ability to enhance transcription of LSR. Thus, in preferred embodiments, addition of the zinc finger protein preferably significantly increases LSR transcription, or significantly increases LSR translation.

By "significantly increases LSR transcription" is meant that the level of transcription following addition of the zinc finger protein is preferably increased at least 2-fold, more preferably at least and most preferably at least 10-fold compared to the level RNA prior to the addition of the zinc finger protein. The assay used may be a Northern blot, or any other assay that measures RNA expression. Alternatively, if the starting level of RNA transcription is below the level of detection by the assay used, "significantly increases LSR transcription" may mean that the level of transcription of LSR may become detectable on the addition of the zinc finger binding protein.

Similarly, by "significantly increases LSR translation" is meant that the level of translation following addition of the zinc finger protein is preferably increased at least 2-fold, more preferably at least 5-fold, and most preferably at least 10-fold compared to the level of translation prior to the addition of the zinc finger protein. The assay used may be a Western blot, or dot blot, or other type of immunoassay for example, or any other assay known in the art to be used to measure or detect the presence of proteins. Alternatively, if the starting level of LSR protein is below the level of detection by the assay used, "significantly increases LSR translation" may mean that LSR protein may become detectable after the addition of the zinc finger binding protein.

These zinc finger proteins can be useful in vivo in gene surgery in which transcription of endogenous LSR is enhanced as part of the treatment for an obesity-related disease or disorder.

This can be envisioned in a situation where higher levels of the LSR protein are thought to be advantageous for the patient clinically. For example, increased expression of LSR could be advantageous when the LSR gene is normal, but is expressed at lower than normal levels, or when it is expressed at normal levels, but does not function as efficiently as it should in clearing triglycerides from the bloodstream, or when some other abnormality results in abnormally high levels of triglycerides and an increased amount of LSR protein is necessary to clear them.

WO 01/21647 PCT/IB00/01470 28 In a further alternative embodiment of the invention, zinc finger proteins are designed to bind to any sequence of 18 or more contiguous base pairs of LSR mRNA and thereby inhibit translation of LSR. In preferred embodiments, expression of all three forms of LSR are inhibited by the zinc finger protein. In an alternative embodiment, zinc finger proteins are designed to specifically inhibit expression of the LSR a, or p subunit individually, or to inhibit both the LSR a and a' subunits. All three forms of LSR can be inhibited by zinc finger proteins targeted to mRNA fragments transcribed from exons one through 3 and exon 6 to the end. The a subunit can be targeted with zinc finger proteins designed to bind in exon 4. The a' subunit can be targeted with zinc finger proteins designed to bind to the splice site between exon 3 and exon 5. The p subunit can be targeted with zinc finger proteins designed to bind to the splice site between exon 3 and exon 6. Both the a and a' subunits can be targeted with zinc finger proteins designed to bind to exon These zinc finger proteins would be useful for many of the uses previously described for zinc finger proteins binding to and inhibiting or increasing transcription ofLSR DNA.

Similarly the definitions for inhibiting or increasing LSR transcription and tests for the desired zinc finger proteins and methods for designing and making them would be as previously described. In addition, for all of the zinc fingers described, it should be remembered that the system can be further controlled by addition of a small molecule control system (for example the Tet-responsive system, or RU486, or ecdysone) to the cell. This allows greater control/greater finesse for an in vitro assay system, in particular, but can be used in vivo as well. The basic idea is to provide the zinc finger with part of the Tet system integrated upstream such that transcription of the zinc finger protein can be regulated by the addition of an outside element, for example Dox or Tc. These methods are well-known to those in the art.

I. Polynuclcotides Encoding Zinc Finger Polypcptides of the Invention The invention also features polynucleotides that encode the zinc finger polypeptides of the invention described above. In one method of identifying the desired zinc finger polypeptides of the invention, libraries are screened (panned) for those clones expressing a zinc finger protein that binds to the desired nucleotide sequence. Frequently, multiple clones are identified that express zinc finger proteins that bind to the nucleotide sequence. All the variant pnlynucleotides that code for the zinc finger polypeptide(s) that bind to the desired sequence are also part of the present invention.

Variants of polynucleotides, as the term is used here, are polynucleotides whose sequence differs from a reference polynucleotide; in this case a reference polynucleotide is the polynucleotide that is ultimately chosen to be used. Thus, the variant of the polynucleotide would frequently be the result of mutagenesis techniques as described in WO 98/54311.

WO 01/21647 PCT/IB00/01470 29 Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.

Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. Alterations in the zinc finger polypeptide coding regions of the invention may produce conservative or non-conservative amino acid substitutions, deletions or additions in the encoded protein. Preferably, the nucleotide substitutions result in non-conservative amino acid changes and more preferably in conservative amino acid changes in the encoded polypeptide.

In cases where the nucleotide substitutions result in one or more amino acid changes, preferred zinc finger polypeptides include those that retain the same activities and activity levels as the zinc finger polypeptide encoded by the reference polynucleotide sequence, as well as those where the level of one or more activities is increased, and alternatively where the level of one or more activities is decreased or even absent. Zinc finger polypeptide activities of the invention and methods for testing are described above.

A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part, but not all, of a given nucleotide sequence, preferably the nucleotide sequence encoding a zinc finger polypeptide, and variants thereof, as described above, and the complements of these polynucleotides. Such fragments may be "free-standing", i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger polynucleotide of which they form a part or region. However, several fragments may be comprised within a single larger polynucleotide. Optionally, such fragments may consist of a contiguous span that ranges in length from 8, 10, 12, 15, 18 or 20 to 25, 35, 40, 50, or 60 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, 50, or 60 nucleotides in length.

IV. Chimeric Oligonucleotides of the Invention Chimeraplasty is a technique used to change the nucleotide sequence of DNA of cells and of animals (Science 285 :316-318 (1999)). It can be used to create or to correct mutations, usually point mutations, that have an effect on the protein coding sequence. The technique relies on hybrid molecules of DNA and RNA called chimeras that contain DNA with a mutation in its sequence (compared to the target sequence in the cell) flanked by RNA that perfectly mirrors the flanking target gene sequence. The target gene sequence is thought to be modified through the action of the cell's DNA repair machinery as a result of the pairing of the target DNA with the chimera containing the mutated sequence.

WO 01/21647 PCT/IB00/01470 In the present invention, the advantages to using chimeraplasty to modify LSR include ease of creating cells lacking LSR polypeptides for use in assays or gene surgery; (2) specifically blocking production of the a subunit or the a and a' subunits for use in assays or in gene surgery; and the ability to correct defects in the LSR gene in cells in vitro and in vivo for use in gene surgery. Chimeraplasty has been shown to be effective for correcting (or creating) mutations in cells in vitro and in vivo in animals (Cole-Strauss, et al. Science 273 13861389 (1996) Alexeev and Yoon Nature Biotechnology 16 1343-1346(1998) Kren et al Nature Medecine 4 285-290 (1998) Yoon et al Proc Natl. Acad. Sci. USA 93 2071-2076 (1996) Xiang et al J Mol Med 75 829-825 1997), hereby encorporated by reference herein in their entirety including any figures, drawings, or tables). Chimeraplasty is particularly useful in cases of point mutations that need to be corrected to allow either expression or function of the protein.

Chimeraplasty apparently works through the cell's own DNA repair system to correct the targeted gene. Although the gene is not corrected in 100% of the cells following transfection in vitro or introduction into the animal in vivo, the genes in enough of the cells have been found to be changed to permit a clinically detectable change. This could, in fact, be beneficial in the LSR system where it is unlikely that you would ever want to completely prevent LSR expression.

However, reduction in LSR expression might be advantageous in some obesity-related diseases and disorders. In particular, specific reduction in any one or more of the a, or p subunits could be advantageous.

The invention features a chimeric oligonucleotide, comprising at least 9 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, wherein said at least 9 contiguous nucleotides comprise at least one amino acid codon selected from the group consisting of TTA, TIG, TCA, TCG, TAU, TAC, TGT, TGC, TGG, CAA, CAG, AGA, GAA, GAG, and GGA, and wherein a point mutation is present in said codon such that said codon is a stop codon. In preferred embodiments, the sequence is selected from the group consisting of Exon 1, Exon 4 and Exon 5 from SEQ ID NO 1 and homologous sequences from mouse or rat, preferably mouse.

Another embodiment of the invention features chimeraplast LSR polynucleotides, where the polynucleotide comprises at least 7 (preferably at least 13, more preferably at least 25, most preferably at least 35 nucleotides of the LSR gene (or its complement), and where the DNA portion of the chimera comprises a point mutation such that instead of coding for an amino acid, it now codes for a termination codon. Thus, substitutiton of this nucleotide for the nucleotide present in the endogenous LSR gene, results in a stop codon being created at the site. The other nucleotides present in both the DNA and RNA portions of the chimera are 100% complementary to the flanking regions of the endogenous LSR gene. The DNA portion of the chimera is at least WO 01/21647 PCT/IB00/01470 31 3 consecutive nucleotides in length, preferably at least 5 consecutive nucleotides in length, optionally at least 7 or at least 11 nucleotides in length. The point mutation is preferably the middle nucleotide (n alternatively n+l, or n-1 less preferably n+2, or n-2 n+3, or n-3, etc.) of the DNA part of the chimera when the DNA portion has an odd number of nucleotides (AGnCT, AnGCT, AGCnT, for example), or the n+l or n-I positions (less preferably n+2, or n-2 n+3, or n-3, etc.) when the sequence has an even number of nucleotides (AnCT, AcnT, for example).

The RNA portion of the chimera is at least 4 consecutive nucleotides in length, preferably at least 10 consecutive nucleotides in length, more preferably at least 20 consecutive nucleotides in length, and most preferably at least 30 consecutive nucleotides in length. The RNA portion of the chimera flanks the DNA portion of the chimera, preferably with an equal number of nucleotides on each side of the DNA sequence (x when the number on RNA residues is even), less preferably with x+ on the upstream side and x-1 on the downstream side or alternatively x+1 on the downstream side and x-1 on the upstream side even less preferably with x+2 on the upstream side and x-2 on the downstream side or alternatively x+2 on the downstream side and x-2 on the upstream side, and so on. Similarly, when the number of RNA residues is odd, there are either x+1 on the upstream side and x-I on the downstream side or alternatively x+1 on the downstream side and x-l on the upstream side of the DNA; less preferably there are x+2 on the upstream side and x-2 on the downstream side or alternatively x+2 on the downstream side and x-2 on the upstream side, and so on. In some cases, particularly when the point mutation is not in the center of the DNA part of the chimera, the number of residues of RNA flanking the DNA is preferably not equal on both sides. In some cases it is preferred that there are more RNA residues on one side than the other so as to have the point mutation be located at the center of the chimera, or at least n+l or n-l from the center of the chimera, less preferably n+2, or n-2 from the center, etc. Sequences that encode stop codons include TAA, TAG, and TGA. Therefore, sequences encoding the amino acids leucine (TTA or TTG), serine (TCA or TCG), tyrosine (TAU or TAC), cysteine (TGT or TGC), tryptophan (TGG), glutamine (CAA or CAG), arginine (AGA), glutamate (GAA or GAG), or glycine (GGA), for example, can be changed to one of the stop codons by a single polynucleotide exchange. The preferred stop codon is TGA. The exact design of the chimeras will depend on the particular sequence to be mutated, but guidance has been given in the papers listed above and in the Examples herein. In general, however, the sequence should be at least 14 nucleotides in length (preferably 18, more preferably 25, most preferably 30) to ensure specificity to the desired sequence. Preferably, the amino acid to be mutated to a termination codon is located at the 5' end of the coding sequence, preferably within the first exon, and preferably is the first amino acid that can be mutated in this way after the first ATG or most preferably the second ATG. Amino acids to be mutated to stop all LSR expression should not be selected from Exon 4 or Exon 5, since exon 4 is not present in the a' subunit, and WO 01/21647 PCT/IB00/01470 32 neither Exon 4 nor Exon 5 is present in the P subunit. The success of a chimeraplast in preventing LSR expression can be tested using the techniques described herein, to include screens for the presence of the mRNA by Northern blot, for example, and for the protein by Western blot, for example.

Alternatively, in some preferred embodiments it is preferable to stop expression of the LSR a subunit only. To do this, the amino acid to be mutated is preferably located in Exon 4 of LSR, since this Exon is not present in the a' or P subunits. In other preferred embodiments it is preferable to prevent expression of both a and a' subunits, but not the P subunit. To do this, the amino acid to be mutated is preferably located in Exon 5 of LSR, since this exon is present in both a and a' subunits, but not the P subunit.

In another embodiment, the invention feaures chimeraplast LSR polynucleotides, where the polynucleotide comprises at least 7 (preferably at least 13, more preferably at least 25, most preferably at least 35 nucleotides of the LSR gene (or its complement), and where the DNA portion of the chimera comprises one of the alleles of the single nucleotide polymorphisms (SNPs) described in U.S. Provisional Application No. 60/119, 592, entitled Polymorphic Markers of the LSR Gene by Blumenfeld, Bougueleret, and Bihain, filed February 10, 1999 and indicated in Table A. Preferably, the SNP's are selected from the group consisting of Al, A2, A3, A4, A5, A6, A7, A8, A9, A10, Al 1, A12, A13, A14, A15, A16, A17, A18, A19, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, AND A32. The SNPs may be in either coding or non-coding regions of the LSR gene. Some SNPs in the coding region result in amino acid changes that may affect the activity of LSR. However, the majority of the SNPs do not code for amino acid changes. These nucleotide changes can also modulate the activity of LSR in a variety of ways, for example by interfering with the binding of a regulatory molecule that influences the splicing of the introns, particularly where there is differential splicing depending on the subunit to be expressed or by affecting the binding of promoters or the function of other regulatory sequences in the 5' and 3' regions of the gene. Changes in the expression of various subunits, or the levels of expression of LSR in general, can have profound effects on the obesity of patients.

VI. Recombinant Vectors of the Invention The term "vector" is used herein to designate either a circular or a linear DNA or RNA molecule, that is either double-stranded or single-stranded, and that comprises at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.

The present invention relates to recombinant vectors comprising any one of the polynucleotides described herein.

WO 01/21647 PCT/IB00/01470 33 The present invention encompasses a family of recombinant vectors that comprise polynucleotides encoding leptin polypeptides of the invention, polynucleotides encoding zinc finger proteins of the invention, and chimeraplastic polynucleotides of the invention as described herein.

In a first preferred embodiment, a recombinant vector of the invention is used to amplify the inserted polynucleotide in a suitable cell host, this polynucleotide being amplified every time that the recombinant vector replicates. The inserted polynucleotide can be one that encodes leptin polypeptides of the invention or zinc finger polypeptides of the invention, or a chimeraplast polynucleotide.

A second preferred embodiment of the recombinant vectors according to the invention, consists of expression vectors comprising either a polynucleotide encoding leptin polypeptides of the invention or zinc finger proteins of the invention, or both. Within certain embodiments, expression vectors are employed to express a leptin polypeptide of the invention, preferably a modified leptin polypeptide described in the present invention, which can be then purified and, for example, be used in screening assays or as a treatment for obesity-related diseases. In other embodiments, expression vectors are employed to express a zinc finger protein of the invention, preferably one that inhibits LSR expression or expression of specific subunits of LSR as described in the present invention, which can be then purified and, for example, be used in screening assays or as a treatment for obesity-related diseases. In other embodiments, the expression vectors are used for constructing transgenic animals and also for gene surgery, in particular, expression vectors containing a polynucleotide encoding zinc finger proteins of the invention.

Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources, that drive expression of the genes of interest in host cells. Dominant drug selection markers for establishing permanent, stable, cell clones expressing the products are generally included in the expression vectors of the invention, as they are elements that link expression of the drug selection markers to expression of the polypeptide.

More particularly, the present invention relates to expression vectors which include nucleic acids encoding a leptin polypeptide fragment of the invention, or a modified leptin polypeptide as described herein, or variants or fragments thereof, under the control of a regulatory sequence selected among the leptin regulatory polynucleotides, or alternatively under the control of an exogenous regulatory sequence. The present also relates to expression vectors which include nucleic acids encoding a zinc finger polypeptide of the invention, or a modified zinc finger polypeptide as described herein, or variants or fragments thereof, under the control of an exogenous regulatory sequence.

WO 01/21647 PCT/IB00/01470 34 Consequently, preferred expression vectors of the invention are selected from the group consisting of: a leptin regulatory sequence and driving the expression of a coding polynucleotide operably linked thereto; a leptin polypeptide coding sequence of the invention, operably linked to regulatory sequences allowing its expression in a suitable cell host and/or host organism. Other preferred expression vectors of the invention comprise a zinc finger polypeptide coding sequence of the invention, operably linked to regulatory sequences allowing its expression in a suitable cell host and/or host organism.

Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.

1) General features of the expression vectors of the invention A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, or even a linear DNA molecule which may consist of a chromosomal, nonchromosomal, semi-synthetic or synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of: a genetic clement or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about to 300 bp in length that act on the promoter to increase the transcription.

a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in and appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a Nterminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable WO 01/21647 PCT/IB00/01470 promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5'-flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

2) Regulatory elements Promoters The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.

A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.

Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter, or the plO protein promoter from baculovirus (Kit Novagen) (Smith et al. (1983) Mol. Cell. Biol.

3:2156-2165.; O'Reilly et al., 1992, Baculovirus expression vectors a Laboratory Manual. W.H.

Freeman and Co., New York the lambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilled in the field of genetic egineering. For example, one may refer to the book of(Sambrook, Fritsch, and T. Maniatis. (1989), Molecular Cloning: A Laboratory Manual. 2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) )or also to the procedures described by (Fuller S.

WO 01/21647 PCT/IB00/01470 36 A. et al. (1996) Immunology in Current Protocols in Molecular Biology, Ausubel et al., Eds, John Wiley Sons, Inc., USA).

Other regulatory elements Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.

Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

The vector containing the appropriate DNA sequence as described above, more preferably LSR gene inhibitory or activating polynucleotide, a polynucleotide encoding a leptin polypeptide or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.

3) Selectable markers Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or zeocin, hygromycin or neomycin resistance for eukaryotic cell culture, TRPI for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli, or levan saccharase for mycobacteria, this latter marker being a negative selection marker.

4) Preferred vectors Bacterial vectors As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEMI (Promega Biotec, Madison, WI, USA).

Large numbers of other suitable vectors are known to those of skill in the art, and are commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX 174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNII46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia);.pWLNEO, WO 01/21647 PCT/IB00/01470 37 pSV2CAT, pOG44, pXTI, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE- (QIAexpress).

Baculovirus vectors A suitable vector for the expression polypeptides of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC NOCRL 1711) which is derived from Spodopterafrugiperda.

Other suitable vectors for the expression of a leptin polypeptide in a baculovirus expression system include those described by (Chai H. et al. (1993), Biotechnol. Appl.

Biochem.18:259-273; Vlasak R. et al. (1983), Eur. J. Biochem. 135:123-126; Lenhard T. et al.

(1996), Gene. 169:187-190).

Viral vectors In one specific embodiment, the vector is derived from an adenovirus. Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996) or Ohno et al. (1994). Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin French patent application N° FR-93.05954).

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.

Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).

Other preferred retroviral vectors are those described in Roth J.A. et al. (1996), Nature Medicine.

2(9):985-991 PCT Application No WO 93/25234, PCT Application No WO 94/ 06920, Roux et al., 1989, Proc. Natl Acad. Sci. USA, 86 9079 9083, Julan et al., 1992, J. Gen. Virol., 73 3251 -3255 Neda et al., 1991, J. Biol. Chem., 266 14143 14146.

WO 01/21647 PCT/IB00/01470 38 Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV). The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992, Curr. Topics in Micro.

and Immunol., 158 97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., 1992, Am. J.

Respir. Cell Mol. Biol., 7:349-356; Samulski et al., 1989, J. Virol., 63 3822-3828; McLaughlin B.A. et al. (1996), Am. J. Hum. Genet. 59:561-569. One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.

Delivery of the recombinant vectors In order to effect expression of the polynucleotides of the invention, these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states.

One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.

Several non-viral methods for the transfer ofpolynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al. (1973), Virology. 52:456-457; Chen et al., 1987, Mol.

Cell. Biol., 7 2745-2752;), DEAE-dextran (Gopal, 1985, Mol. Cell. Biol., 5 1188-1190 electroporation (Tur-Kaspa et al. (1986), Mol. Cell. Biol. 6:716-718; Potter et al., 1984, Proc Natl Acad Sci U S A. 81(22):7161-5) direct microinjection (Harland et al., 1985, J. Cell. Biol., 101:1094-1095) DNA-loaded liposomes (Nicolau et al., 1982, Biochim. Biophys. Acta, 721:185- 190; Fraley et al., 1979, Proc. Natl. Acad. Sci. USA, 76 3348-3352 and receptor-mediate transfection (Wu and Wu, 1987, J. Biol. Chem, 262 4429-4432; Wu and Wu,1988, Biochemistry, 27:887-892). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.

WO 01/21647 PCT/1B00/01470 39 One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect. This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.

Compositions for use in vitro and in vivo comprising a "naked" polynucleotide are described in PCT application No. WO 90/11092 (Vical Inc.) and also in PCT application No.

WO 95/11307 (Institut Pasteur, INSERM, Universit6 d'Ottawa) as well as in the articles of Tacson et al. (1996) Nature Medicine. 2(8):888-892 and Huygen et al. (1996) Nature Medicine.

2(8):893-898.

In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al. (1987) Nature. 327:70-73.

In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, 1991, Targeting of liposomes to hepatocytes, IN Liver Diseases, Targeted diagnosis and therapy using specific receptors and ligands. Wu et al. Eds., Marcel Dekekcr, New York, pp. 87-104; Wong et al., 1980, Gene, 10 87-94; Nicolau C. et al.

(1987), Methods Enzymol. 149:157-76). These liposomes may further be targeted to cells expressing LSR by incorporating leptin, triglycerides, Acrp30, or other known LSR ligands into the liposome membrane.

In a specific embodiment, the invention provides a composition for the in vivo production ofa leptin polypeptide, or a zinc finger protein, described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said polypeptide.

The amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0,1 and 100 ig of the vector in an animal body, preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired leptin polypeptide or the desired WO 01/21647 PCT/IB00/01470 fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.

V. Recombinant Cells of the Invention The invention is in part based on the surprising and unexpected discovery that the different subunits of LSR interact to form at least two very different receptors LSR-lep and LSR-tg. The LSR-lep receptor requires at least In some embodiments a combination with 0 and/or a as well as a' is preferred. The LSR-tg receptor requires a combination of at least a and p. In some embodiments a combination with P and/or a as well as a' is preferred. Based on this novel and unexpected finding, it has become critical to engineer cells lacking endogenous LSR activity/expression as a result of a classical knock-out, chimeraplasty, or zinc finger protein inhibition), and then to re-transfect the subunits of interest in various combinations and at various levels. This will allow not only the study of these receptors in isolation, but also the design of specific inhibitors for the different receptors, and the assessment of what genes may act to regulate or modulate the receptors, or to transmit the intracellular signals from or for each receptor. Although LSR-lep and LSR-tg have been identified, it is possible that other LSR receptors with other activities also exist and can be identified by these methods.

Recombinant cells have been designed that are useful in many situations, including (1) the study of the role of the various LSR components in isolation and together with and without interference from endogenous LSR, as part of an assay system to discover modulators of the leptin/LSR interaction, for example, using known components of the LSR system (and in some cases no endogenous LSR components; see above), and to produce various polypeptides of the invention (see above). To this end, in preferred embodiments, a recombinant cell is transiently, or preferably stably, transfected with one or more LSR subunits selected from the group consisting of a, a' and p. Preferably, the two or more subunits are expressed in pairwise ratios to each other of from 1:1 to 1:5. For example, if a and P are present in a cell, cells with ratios of 1 1 1 1 1 5 4 3 2 as well as 2:3, 3 3 :4,4 3 5 :3, 4 and 5 etc. are preferred. Similar ratios are desired for cells containing a' and P. When all three subunits are present, cells with all possible combinations of ratios are preferred. These are most easily obtained by screening cells (wild-type, transfected. or knockout, for example) for their expression levels of the various subunits. Preferably, the one or more LSR components are a' and P, and preferably the recombinant cells are cultured PLC cells. However, the cells can be selected from any of the cells in the ATCC bank. The LSR polypeptides, the polynucleotides encoding LSR, and the vectors to transfer the polynucleotides encoding LSR WO 01/21647 PCT/IB00/01470 41 between cells and tissues have been described previously (US National phase application No.

09/269,939).

Another object of the invention consists of host cells that have been transformed or transfected with one of the polynucleotides described herein, and more precisely a polynucleotide comprising: a polynucleotide encoding a leptin polypeptide of the invention, or a polynucleotide encoding a zinc finger protein of the invention. These polynucleotides can be present in the same cell or in a different cell, and can be present in cells transiently or stably transfected with any combination of the components of LSR.

In another embodiment, the invention features cells that lack expression of at least one of the LSR subunits. These can be cells identified by screening processes, but they are preferably recombinant cells that have had the gene for LSR knocked-out by traditional techniques well known in the art; a cell in which a polynucleotide encoding a zinc finger protein of the invention has been transfected that either constitutively suppresses the expression of at least one subunit of LSR or whose suppression of LSR can be regulated by the Tet On/Off system, for example; or a cell in which the expression of at least one subunit of LSR has been inhibited as the result of the transfection of chimeric oligonucleotides of the invention.

The invention further features either transiently, or preferably stably, transfecting the LSR knockout cells (or zinc finger protein cells) in which expression of at least one, and in some cases all, of the endogenous LSR subunits has been inhibited (or eliminated), with at least one, preferably at least two, and alternatively three, of the LSR subunits and then selecting/screening for cells expressing the various ratios of subunits as described above. Preferably, P, a or a' alone are transfected, or alternatively a' and P, or a and P together are transfected.

The invention includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as any one of those described in Recombinant Vectors of the Invention Generally, a recombinant host cell of the invention comprises at least one of the polynucleotides or the recombinant vectors of the invention which are described herein, but also includes those cells in which the gene for LSR has been knock-out by traditional recombinant techniques, zinc finger techniques, or using chimeraplast oligonucleotides.

Preferred host cells used as recipients for the recombinant vectors of the invention are the following a) Prokaryotic host cells Escherichia coli strains DH5-a strain), Bacillus subtilis, Salmonella typhimurium, and strains from species like Pseudomonas, Streplomyces and Staphylococcus, and b) Eukaryotic host cells: HeLa cells (ATCC NOCCL2; NOCCL2.1; NOCCL2.2), Cv 1 cells (ATCC NOCCL70), COS cells (ATCC NOCRL1650; NOCRL1651), Sf-9 cells (ATCC WO 01/21647 PCT/IB00/01470 42 N°CRLI711), C127 cells (ATCC No CRL-1804), 3T3 (ATCC No CRL-6361), CHO (ATCC No CCL-61), human kidney 293 (ATCC No 45504; No CRL-1573), BHK (ECACC No 84100501; N° 84111301), PLC cells, HepG2, Hepa 1-6, and Hep3B.

The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by the skilled artisan.

Further, according to the invention, these recombinant cells can be created in vitro or in vivo in an animal, preferably a mammal, most preferably selected from the group consisting of mice, rats, dogs, pigs, sheep, cattle, and primates, not to include humans. Recombinant cells created in vitro can also be later surgically implanted in an animal, for example. Methods to create recombinant cells in vivo in animals are well-known in the art, and are specifically meant to include the techniques associated with chimeraplasty described herein and known in the art, whereby the chimeraplast oligonucleotides are provided to the cells in the animal by the use of liposomes, preferably liposomes that have targeting molecules for cells containing LSR such as LSR binding proteins or ligands, such as apml, Clq, or leptin, for example, in the membrane layer.

VII. Assays for Identifying Modulators of LSR Activity The surprising and unexpected discovery that the different subunits of LSR interact to form at least two very different receptors (LSR-lep and LSR-tg) with different activities has resulted in the necessity of designing novel assays to identify inhibitors for the different LSR receptors. In particular, these assays will preferably utilize the recombinant cells of the invention, that are engineered to lack endogenous LSR activity/expression as a result of a classical knock-out, chimeraplasty, or zinc finger protein inhibition). These cells are then retransfected with the subunits of interest in various combinations and at various levels. Preferred combinations include those that give rise to the LSR-lep receptor that requires at least but may also include combination of a' and P, and the LSR-tg receptor that requires a combination of a and P. Other combinations (and the individual subunits) are also useful to look for other WO 01/21647 PCT/IB00/01470 43 LSR receptor activities and as controls for the activity of compounds (or genes) selected in the other assays.

The invention features methods of screening for one or more compounds that modulate LSR activity in cells, that includes providing potential compounds to be tested to the cells, and where modulation of LSR activity indicates the one or more compounds. In some preferred embodiments, the potential compounds are compounds that have been molecularly designed based on the identified fragment of leptin that binds and activates LSR as described herein.

In a preferred embodiment, the invention features a method for selecting a compound useful for the treatment or prevention of an obesity-related disease or disorder, comprising: contacting a recombinant cell that comprises a zinc finger protein of the invention, or a recombinant vector comprising any of the zinc finger proteins of the invention with a candidate compound; and detecting a result selected from the group consisting of a modulation of an activity of the Lipolysis Stimulated Receptor and modulation of expression of the Lipolysis Stimulated Receptor; as a means for selecting said compound useful for the treatment or prevention of said obesity-related disease or disorder.

In preferred embodiments, said contacting is in the presence of a ligand of said Lipolysis Stimulated Receptor. Preferably, said ligand is selected from the group consisting of cytokine, lipoprotein, free fatty acid, adipoQ (Apml and Acrp30), and Clq, and more preferably said cytokine is leptin. Alternatively, said free fatty acid is oleate. In other preferred embodiments, said leptin is a leptin polypeptide fragment that modulates the activity of LSR, comprising at least 4, but not more than 50 contiguous amino acids of any one of the leptin polypeptide sequences set forth in Figure 13, wherein said at least 4 and not more than 50 contiguous amino acids comprise the leptin fragment central sequence. In other preferred embodiments, said leptin is a variant of a leptin polypeptide fragment that modulates the activity of LSR, consisting of a 22 contiguous amino acid sequence that is at least 75% identical to the leptin fragment variable region of any one of the leptin polypeptide sequences set forth in Figure 13. Optionally, the leptin fragment is any leptin fragment of the invention described herein.

In other preferred embodiments of the invention, said activity is selected from the group consisting of binding of lipoproteins, uptake of lipoproteins, degradation of lipoproteins, binding of leptin, uptake of leptin, and degradation of leptin. Preferably, said modulation of LSR activity is an increase in said activity, and optionally a decrease in said activity. In other preferred embodiments, said expression is on the surface of said cell, and preferably said detecting comprises FACS, more preferably said detecting further comprises antibodies that bind specifically to said LSR, wherein said LSR comprises an amino acid sequence at least homologous to at least one of the sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO:18, WO 01/21647 PCT/IB00/01470 44 and SEQ ID NO:19. In other preferred embodiments, said amino acid sequence is at least 80, 95, or 99 to 100% homologous to at least one of the sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In other preferred embodiments, said antibodies bind specifically to a region of said LSR selected from the group consisting of an amino terminus, a carboxy terminus, a splice site, a cytokine binding site, a fatty acid binding site, a clathrin binding site, an apoprotein ligand binding site, a L/LL motif, a RSRS motif, and a hydrophobic region. Preferably, said cell is selected from the group consisting of PLC, CHO- Kl, Hep3B, and HepG2, although any cell expressing detectable levels of LSR can be used.

Antibodies to LSR and to the various regions of LSR have been extensively described previously in US National application 09/269,939, filed May 28, 1999 and its related PCT application. In addition, specific antibodies to LSR are described in the Examples In preferred embodiments, said candidate compound is selected from the group consisting ofpeptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, and small molecules, and optionally can include leptin mimetics designed by methods of the invention. The compounds may be active in vitro or in vivo. The activity may be increased or decreased the compounds may be antagonists or agonists.

Preferably, said obesity-related diseases and disorders are selected from the group consisting of obesity, anorexia, cachexia, cardiac insufficiency, coronary insufficiency, stroke, hypertenison, atheromatous disease, atherosclerosis, high blood pressure, non-insulin-dependent diabetes, hyperlipidemia, and hyperuricemia. The compounds may also modulate body mass. Most preferably, the diseases include congenital generalized lipodystrophy.

In other highly preferred embodiments of the invention, the cells used in the abovedescribed assays cells have been modified to express none, or a subset, of the LSR subunits.

The recombinant cells containing zinc finger proteins of the invention are also transfected with at least one polynucleotide encoding a LSR polypeptide comprising a sequence at least identical to an amino acid sequence selected from the group consisting of.SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO: 19. Preferably the LSR subunit is stably transfected. Preferably the cell is selected from the group consisting of PLC, CHO-KI, Hep3B, Hepa 1-6, and HepG2. However, other cells available from the ATCC, for example, may also be used. In addition, cells with the endogenous LSR gene knocked out by methods well-known in the are are also expressly contemplated (as an option to the use of the zinc finger proteins of the invention, or to the use of the chimeraplasts of the invention.). Cells, preferably modified cells, are transfected with one or more LSR components that may include one, part, or all, of a, and p, most preferably a' and p. Recombinant cells useful for assays to identify modulators of the leptin-LSR interaction WO 01/21647 PCT/IB00/01470 include those described in the Recombinant Cells of the Invention o. In particular, cells expressing a range of ratios of the subunits are desired, including 1 1 1 1 1 5 :1, 4 3 2 as well as 2:3, 3 3 4 3 5 4 and 5 etc. for a' to 0 or a to 3, or even a to for example. In addition, the various combinations where all three subunits are present in a cell are also envisioned to be useful in assays for modulators of LSR activity.

In highly preferred embodiments of the invention, cells with endogenous LSR activity knocked-out and transfected with the a' alone, or a' and p LSR subunits together are used to screen for modulators of the LSR-leptin interaction. In other preferred embodiments, the a and P LSR subunits are used to screen for modulators of triglyceride-rich lipoprotein binding, uptake, and degradation. Cells with all three LSR subunits are useful to screen for modulators of the effect of leptin binding uptake and degradation on triglyceride-rich lipoprotein binding, uptake and degradation. Similarly, these cells would be useful for screening molecules arising from the active leptin fragment molecular modeling described herien.

IX. Methods for Designing Leptin Polypeptide Fragment Mimetics Following the discovery of the differential results of human and mouse leptin on human and rodent LSR, the region of amino acid sequence sharing the least homology between the two homologs was identified and was found to stimulate rodent and human LSR activity differentially (Examples Identification of the differences between these two highly similar peptides allows the design of small molecule activators or inhibitors of LSR. Methods of determining the differences are well known in the art and include, but are not limited to techniques such as molecular dynamic assays, X-ray crystallography, and NMR. Previously, these kinds of techniques for creating inhibitors/activators of enzymes have been used successfully in the art. Potential small molecule activators/inhibitors designed or identified by these methods can be tested in the assays described herein. Those that function in these assays can then be tested for their effectiveness for treatment of obesity-related disorders and diseases, as described herein, for activity in modulating body mass, and for activity in treating congenital generalized lipodystrophy (Example 14).

The invention features a method of designing mimetics of a leptin fragment that modulates an activity of LSR, comprising: identifying critical interactions between one or more amino acids of said leptin fragment and LSR; designing potential mimetics to comprise said critical interactions; and testing said potential mimetics ability to modulate said activity as a means for designing said mimetics. By designing mimetics as used herein is meant comparing and combining known molecules to obtain a molecule that is able to mimic some or all of the activities modulated by leptin, or to preferentially increase or decrease some of the activities normally modulated by leptin. These activities include, but are not limited to WO 01/21647 PCT/IB00/01470 46 those activities selected from the group consisting of leptin binding, leptin uptake, leptin degradation, triglyceride binding, triglyceride uptake, and trglyceride degradation. The methods of comparing and combining use molecular modeling, X-Ray crystallography and other techniques well-known in the art to identify the critical interactions. These critical interactions include, but are not limited to those selected from the group consisting of hydrogen bonding, covalent bonding, Van der Waals forces, steric hindrances, and hydrophobic interactions. These critical interactions are identified using assays that include, but are not limited to, those selected from the group consisting of NMR, X-ray crystallography, and computer modeling. Preferably the non-leptin compounds that are identified or designed by these means include, but are not limited to, small molecules (molecular weight <500, alternatively between 500 and 1000 MW, or >1,000 MW), peptides, peptide libraries, non-peptide molecules, non-peptide libraries and peptoids.

In preferred embodiments, the leptin fragment to be mimicked consists of the leptin fragment variable region of any one of the leptin polypeptide sequences set forth in Figure 13, preferably the human or mouse sequence, most preferably the human sequence. In other embodiments, the leptin fragment consists of the leptin fragment central sequence of any one of the leptin polypeptide sequences set forth in Figure 13, preferably the human or mouse sequence, most preferably the human sequence.

Methods of studying the structure of enzyme-substrate complexes are well known in the art. X-Ray crystallography allows the determination of the precise three-dimensional positions of most of the atoms in a protein molecule. To do this, a source of x-rays, a protein crystal, and a detector are needed. Obtaining the crystal is necessary because the techniques requires that all the molecules are precisely positioned. Methods to produce crystals are well-known in the art.

X-rays going through the protein crystal are scattered by electrons, thus the amplitude of the wave scattered by an atom is proportional to its number of electrons. The scattered waves then recombine, either reinforcing one another on the film or cancelling each other out, depending on the atomic arrangement. From this information, the image is formed by applying a mathematical relation called a Fourier transform, and from here an electron-density map can be calculated, and then interpreted. The limiting resolution for a protein with a good crystal is typically 2 A.

Two methods important for enzyme-ligand interactions include the difference Fourier method, and production of stable complexes. In the Fourier method, the enzyme is crystallized (in this case LSR) and then the X-ray diffraction of the crystalized protein in solvent is compared with the X-ray diffraction of the crystallized protein in the presence of ligand (in this case the 22 amino acid leptin peptide). Provided that there are no drastic changes in the structure or packing of the protein when it binds the ligand, the structure of the complex can be solved by comparing the diffrences between the diffraction patterns. This allows the electron WO 01/21647 PCT/IB00/01470 47 density of the bound ligand and minor changes in the protein structure to be obtained without starting from scratch.

Alternatively, the X-ray diffraction pattern of a stably bound complex can be used to determine the protein-ligand interactions. Sometimes this is done using an inhibitor of the ligand, but can also be achieved under unreactive conditions such as weakly reactive conditions due to pH conditions, ionic state, or very low temperature, using a chemically modified protein or ligand in which important residues are modified, or under conditions in which the equilibrium conditions are shifted.

X-ray crystallography can be complemented by nuclear magnetic resonance (NMR) spectroscopy, which can reveal the structure ofmacromolecules in solution. Certain atomic nuclei such as hydrogen are intrinsically magnetic. The spinning of the positively charged proton, generates a magnetic moment. This moment can take either of two orientations when an external magnetic field is applied. The flow of electrons around a magnetic nucleus generates a small local magnetic field that opposes the external field. Under different environments the energy is absorbed at different resonance frequencies, an effect termed a chemical shift.

Comparison of the shifts and spin-spin couplings, as well as the nuclear Overhauser effect (NOESY spectra) leads to the identification of pairs of protons that are less than 5A apart.

Overlapping peaks in NOESY spectra can be further resolved by obtaining NMR spectra of proteins labelled with 15N and 13C (multidimensional NMR spectroscopy). Typically highly concentrated solutions of proteins are required (1 mM or 15 mg/ml for a 15 kd protein) and the size is generally limited to 30 kd.

Molecular modelling by computer is also used extensively to augment, supplement and integrate the information gained by X-Ray crystallography, NMR, EPR and other techniques. In particular, computer programs such as DOCK allow the prediction, identification, and three-D testing of inhibitors and activators of enzymes. This methodology has been used successfully previously to identify inhibitors. Basically, using the information gained from X-ray crystallography, NMR, and direct modelling, computer programs can now predict the residues that are important for the ligand-protein interactions and can predict structures that can perform the same interactions and test compounds proposed to be able to perform the same interactions.

Through this interplay, molecules can be designed and identified to activate LSR in the manner of the leptin peptide, or to inhibit this interaction. The advantages to designing a molecule in this way include the ability to use compounds that the body cannot metabolize as rapidly as a peptide, that are less expensive to make, and that hopefully lack any unwanted leptin-associated side-effects.

WO 01/21647 PCT/IB00/01470 48 X. Pharmaceutical Compositions of the Invention The identified compounds can be administered to a mammal, including a human patient, alone or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s) at therapeutically effective doses to treat or ameliorate a variety of disorders associated with lipid metabolism. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms of obesity-related diseases or disorders as determined by the methods described herein. Thus, a therapeutically effective dosage of a leptin polypeptide fragment of the invention, or an antagonist or agonist of the leptin-LSR interaction, or a leptin fragment mimetic designed from molecular modeling studies, will be that dosage of the compound that is adequate to promote reduced or increased triglyceride-rich lipoprotein levels following a high-fat meal and that will promote weight loss or weight gain with continued periodic use or administration. Similarly, a therapeutically effective dosage of a chimeric oligonucleotide of the invention or a polynucleotide encoding a zinc finger protein of the invention will be that dosage of the compound that is adequate to increase or reduce triglyceride-rich lipoprotein levels following a high-fat meal and that will promote weight loss or weight gain with continued periodic use or administration. Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

Additional aspects of the invention feature the use of the compounds, chimeric oligonucleotides and zinc fingers, described throughout the application as modulators of LSR activity in the making of medicaments for the treatment of diseases and disorders described in the following section as well as throughout the application. These diseases or disorders include, but are not limited to, anorexia, cachexia, AIDS-related weight loss, neoplasia-related weight loss, or obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type I diabetes, ocular lesions caused by microangiopathy in obese individuals with Type II diabetes, and renal lesions caused by microangiopathy in obese individuals with Type II diabetes. Modulators of body mass are also expressly included, as are compounds (such as the leptin fragments of the invention) for treating congenital generalized lipodystrophy.

WO 01/21647 PCT/IB00/01470 49 Routes of Administration.

Suitable routes of administration include oral, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections. A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering the compound over an extended period of time. Sustained release formulations of the invented medicaments particularly are contemplated.

Composition/Formulation Pharmaceutical compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include a pharmaceutically acceptable carmer and at least one polypeptide that is a leptin polypeptide of the invention. In addition to medicaments that include leptin polypeptides of the invention, non-protein compounds designed based on molecular modeling of the active leptin polypeptide of the invention also will find utility as modulators of LSR activity, both in vitro and in vivo. Further, antagonists and agonists of the leptin-LSR interaction, including leptin and/or triglyceride-rich lipoprotein binding, uptake and degradation will also find utility in modulating LSR activity and/or stimulating a reduction of plasma lipoproteins and/or promoting weight loss.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.

Such penetrants are generally known in the art.

Pharmaceutical preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

WO 01/21647 PCT/IB00/01470 For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, carbon dioxide. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount Capsules and cartridges of, gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained release materials have been established and are well known by those skilled in the art.

Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or cxcipients. Examples of such carriers or excipients include but are not limited to calcium WO 01/21647 PCT/IB00/01470 51 carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.

Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to effect enhanced or inhibited LSR activity in an in vitro system. Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and Compounds that exhibit high therapeutic indices are preferred.

The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDS0, with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, Fingl et al., 1975, in "The Pharmacological Basis ofTherapeutics", Ch. 1).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain the LSR modulating effects. Dosages necessary to achieve the LSR modulating effect will depend on individual characteristics and route of administration.

Dosage intervals can also be determined using the value for the minimum effective concentration. Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; WO 01/21647 PCT/IB00/01470 52 and most preferably between 50-90/o. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

A preferred dosage range for the amount of a leptin polypeptide of the invention, or compound designed based on its molecular modeling, or an antagonist or agonist of its activity with LSR, that can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoprotcins, range from 0.1 50 mg/kg body mass. A more preferred dosage range is from 0.2 25 mg/kg. A still more preferred dosage range is from 1.0 -20 mg/kg, while the most preferred range is from 2.0 10 mg/kg. Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.

XI. Methods of Preventing or Treating Obesity-related Diseases and Disorders A method of preventing or treating obesity-related diseases and disorders comprising providing a patient in need of such treatment with a leptin polypeptide fragment or a leptin mimetic of the invention. Preferably, the leptin polypeptide fragment or mimetic modulates the activity of LSR, more preferably increases the activity of LSR, and optionally decreases the activity of LSR either in vitro or in vivo. Preferably the leptin polypeptide fragment or mimetic is provided to the patient in a pharmaceutical composition that is preferably taken orally. Preferably the patient is a mammal, and most preferably a human. In preferred embodiments, the obesity-related disease or disorder is selected from the group consisting of anorexia, cachexia, AIDS-related weight loss, neoplasia-related weight loss, or obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese individuals with Type 1 diabetes, and renal lesions caused by microangiopathy in obese individuals with Type II diabetes. Modulators of body mass (weight gain or loss) are also expressly included, as are compounds (such as the leptin fragments of the invention) for treating congenital generalized lipodystrophy.

Alternatively, the invention features a method of preventing or treating obesity-related diseases and disorders comprising providing a patient in need of such treatment with a compound identified by assays of the invention. Preferably these compounds antagonize or agonize the interaction of leptin and LSR. In other embodiments, the compounds are those created as a result of the molecular modeling of the active leptin polypeptide and are nonpeptide mimetics that function in the same manner as the active leptin polypeptide of the WO 01/21647 PCT/IB00/01470 53 invention. Preferably, the compound is provided to the patient in a pharmaceutical composition that is preferably taken orally. Preferably the patient is a mammal, and most preferably a human. In preferred embodiments, the obesity-related disease or disorder is selected from the group consisting of anorexia, cachexia, AIDS-related weight loss, neoplasiarelated weight loss, or obesity-related atherosclerosis, obesity-related insulin resistance, obesityrelated hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese individuals with Type I diabetes, and renal lesions caused by microangiopathy in obese individuals with Type II diabetes. Modulators of body mass are also expressly included, as are compounds (such as the leptin fragments of the invention) for treating congenital generalized lipodystrophy.

The invention also features a method for treating or preventing obesity-related diseases or disorders involving gene surgery. To this end, it is advantageous in some conditions to either express more or less LSR, or alternatively to express more or less of one or more LSR subunits.

Using the methods described herein, it is possible to modulate the levels of expression of LSR, or of some LSR subunits using zinc finger polypeptides of the invention or chimeric oligonucleotides of the invention. Preferably, the zinc finger polypeptides are provided to an individual in need of such treatment by polynucleotides encoding the zinc finger polypeptides of the invention. Preferably the zinc finger polynucleotides of the invention are present in a recombinant vector, preferably a retroviral vector, more preferably AAV. Preferably the chimeric oligonucleotides are provided to a patient in need of such treatment using liposomes.

Preferably the liposomes are constructed such that molecules targeting the liposomes to cells containing LSR are present in the membrane. Preferably the molecules include leptin, apml, and Clq, for example. Alternatively they may have compounds that target them to the liver, such as glucose, for example, or alternatively to adipose tissue. Preferably the patient is a mammal and the obesity-related disease or disorder is selected from the group consisting of anorexia, cachexia, AIDS-related weight loss, neoplasia-related weight loss, or obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese individuals with Type II diabetes, and renal lesions caused by microangiopathy in obese individuals with Type II diabetes. Modulators of body mass are also expressly included, as are compounds (such as the leptin fragments of the invention) for treating congenital generalized lipodystrophy.

Still another aspect of the invention relates to the use of chimeric oligonucleotides to specifically alter single nucleotide polymorphisms in a patient in need of such treatment. Single polymorphisms associated with the LSR gene and with obesity have been described in U.S.

provisional application No. 60/119,592, entitled "Polymorphic Markers of the LSR gene" by WO 01/21647 PCT/IB00/01470 54 Blumenfeld et al, filed February 10, 1999, shown in Table A. In one embodiment, this medicament can be used for reducing food intake in obese individuals, reducing the levels of free fatty acids in obese individuals, decreasing the body weight of obese individuals, or treating an obesity related condition selected from the group consisting of obesity-related atherosclerosis, obesity-related insulin resistance, obesity-related hypertension, microangiopathic lesions resulting from obesity-related Type II diabetes, ocular lesions caused by microangiopathy in obese individuals with Type II diabetes, and renal lesions caused by microangiopathy in obese individuals with Type I diabetes. Modulators of body mass are also expressly included, as are compounds (such as the leptin fragments of the invention) for treating congenital generalized lipodystrophy.

Table A Biallelic Marker Localization Polymorphism Frequency AA Marker Position Marker Name In LSR Gene Of Allele 2 Change 99-14410/373 Al 5'regulatory Allele 1: C 373 of region Allele 2: T SEQ ID No 2 99-14424/353 A2 5'regulatory Allele 1: A 353 of region Allele 2: G SEQ ID No 3 99-14418/322 A3 5 'regulatory Allele 1: A 322 of region Allele 2: G SEQ ID No 4 99-14417/126 A4 5'regulatory Allele 1: C 126 of region Allele 2: T SEQ ID No 99-14417/334 A5 5'regulatory Allele 1: C 334 of region Allele 2: T SEQ ID No 99-14415/106 A6 5'regulatory Allele 1: C 106 of region Allele 2: T SEQ ID No 6 99-14413/250 A7 5'regulatory Allele 1: A 250 of region Allele 2: C SEQ ID No 7 99-14413/383 A8 5'regulatory Allele 1: G 383 of region Allele 2: T SEQ ID No 7 99-4575/226 A9 5'regulatory Allele 1: T 25% 226 of region Allele 2: C SEQ ID No 8 9-19/148 A10 5'regulatory Allele 1: C 15% 1243 of region Allele 2: T SEQ ID No 1 9-19/307 All S'regulatory Allele 1: A 12% 1401 of WO 01/21647 WO 0121647PCT/IBOO/01470 region Allele 2: T SEQ ID No 1 9-19/442 A12 S regulatory Allele 1: C 1535 of region Allele 2: Del C SEQ ED No 1 9-20/187 A13 S'regulaiory Allele 1: A 1788 of region Allele 2: C SEQ IDNol1 9-1/308 A;14 Intron 1 Allele 1: C 24% 2391 of Allele 2: G SEQ IDNol1 9-3/324 A15 Exon2 Allele 1: C 29%/ 3778 of Allele 2: T SEQ IDNoI1; 595 of SEQ ID Nos 13, 15, and 17 99-14419/424 Al16 Intron 2 Allele 1: C 22% 4498 of Allele 2: A SEQ IDNoI1 9-24/260 Al17 !ntron 3 Allele 1: A 35% 15007 of Allele 2: G SEQ IDNoI1 9-24/486 A18 Intron 4 Allele 1: G 15% 15233 of Allele 2: A SEQ IDNoI1 9-6/187 A19 Exon 5 Allele1: C 1% 15826 of Allele 2: T SEQ IDNoI1; 940 of SEQ ID No 13; 883 of SEQ MD No 9-7/1 48 A20 Inlron 5 Allele 1: G 35% 19567 of Allele 2: A SEQ IDNo 1 9-7/325 A21 Exon 6 Allele 1: G 14% S-*N 19744 of Allele 2: A SEQ ID No 1; 1191 of SEQ ID No 13; 1134 of SEQ ID No I 987 of SEQ ID No 17 9-7/367 A22 Intron 6 Allele 1: A 19786 of WO 01/21647 WO 0121647PCT/IBO0101470 Allele 2: C SEQ ID No 1 9-9/246 A23 Exon 8 Allele 1: C 50.5% P4R 20158 of Allele 2: G SEQTD Nol1; 1362 of SEQ ID No 13; 1305 of SEQ ID No 1158 of SEQ ID No 17 LSRX9-BM A24 Exon 9 Allele 1: AGG Del 26% Del R 20595 of (17-1/240) Allele 2: Del SEQ ID No 1; AGO 1658 of SEQ ID No 13; 1601 of SEQ ID No 1454 of SEQ ID No 17 LSRXIO-BM A25 Exonl10 AlleleI: T 21108 of Allele 2: G SEQ IDNoI1; 2079 of SEQ ID No 13; 2022 of SEQ ID No 1875 of SFQ ID No 17 99-4580/296 A26 3'regulatory Allele I :A 24% 296 of region Allele 2 G SEQ ID No 9 99-4567/424 A27 3'regulatory Allele I1: C 424 of region Allele 2 T SEQ ID No 99-14420/477 A28 3'regulatory Allele I G 477 of region Allele 2: T SEQ IDNol11 99-4582/62 A29 3'regulatory Allele I A 62 of region Allele 2: G SEQ H) No 12 99-4582/359 A30 3'regulatory Allele I G 24% 359 of region Allele 2. T1 SEQ ID No 12 WO 01/21647 PCT/IB00/01470 17-2/297 A31 5' regulatory Allele 1 C 48% 818 of SEQ ID No region Allele 2: G 1 9-19/256 A32 5' regulatory Allele 1 A 1374 of SEQ ID regio Allele 2: G No I XII: Methods for Selecting Genes that Modulate LSR Expression Another aspect of the invention features a method for selecting for genes that modulate the expression of LSR. This method relies on the use of a retroviral vector to provide cells of choice (those that express LSR naturally or recombinantly, and in any combination of subunits and subunit levels) with genes of interest at a moderate level. By a moderate level is meant a level that is intermediary between high and low, as based on the level of expression of GFP.

Neither high nor low expression is desired since low levels might result in undetectable effects on LSR activity and high levels might co-opt the use of the cell machinery such that LSR isn't made simply for this reason. These moderate levels are easily detected and selected for by FACS analysis as described in the Examples. This method also relies on the use of FACS to detect changes in the activity of LSR as judged by detecting the expression of LSR, or LSR subunits on the surface of the cells, or alternatively intracellularly as well. This can be done by using two antibodies that bind specifically to different regions of LSR, for example the 81B and 93A antibodies.

Thus, in a preferred embodiment, the invention features a method of selecting for genes that modulate an activity of the Lipolysis Stimulated Receptor, comprising providing a retroviral gene library to cells that express said Lipolysis Stimulated Receptor; contacting said cells with a ligand of said Lipolysis Stimulated Receptor; and detecting a change in said activity of the Lipolysis Stimulated Receptor as a means for selecting for said genes. Preferably, said retroviral gene library comprises a cDNA library from tissues selected from the group consisting of liver, brain, muscle, and adipose, and preferably further comprises a detectable marker protein selected from the group consisting of GFP, truncated CD2, and truncated CD4. In preferred embodiments, the method further comprises selecting said cells transfected with the retroviral vector for moderate expression of GFP. Preferably, said selecting of cells is by FACS.

In other preferred embodiments, said ligand is selected from the group consisting of cytokine, free fatty acid, lipoprotein, adipoQ (Acrp30, Apml), and Clq, and preferably said cytokine is leptin. Preferably said free fatty acid is oleate. More preferably, said leptin is a leptin polypeptide fragment that modulates the activity of LSR, comprising at least 4, but not more than 50 contiguous amino acids of any one of the leptin polypeptide sequences set forth in Figure 13, wherein said at least 4 and not more than 50 contiguous amino acids comprise the WO 01/21647 PCT/IB00/01470 58 leptin fragment central sequence. Optionally, said leptin is a variant of a leptin polypeptide fragment that modulates the activity of LSR, consisting of a 22 contiguous amino acid sequence that is at least 75% identical to the leptin fragment variable region of any one of the leptin polypeptide sequences set forth in Figure 13.

In other preferred embodiments, said detecting a change in said activity is by FACS, preferably said detecting further comprises fluorescent antibodies that bind specifically to said LSR, wherein said LSR comprises an amino acid sequence at least 75% homologous to at least one of the sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. More preferably, said antibodies bind specifically to a region of said LSR selected from the group consisting of an amino terminus, a carboxy terminus, a splice site, a cytokine binding site, a fatty acid binding site, a clathrin binding site, an apoprotein ligand binding site, a LI/LL motif, a RSRS motif, and a hydrophobic region.

Antibodies to LSR and to the various regions of LSR have been extensively described previously in US National application 09/269,939, filed May 28, 1999 and its related PCT application. In addition, specific antibodies to LSR are described in the Examples In other preferred embodiments said cell is selected from the group consisting of PLC, CHO-K Hep3B, and HepG2. In some of these embodiments, said cell has had the endogenous LSR activity inhibited by either a traditional knockout of the gene encoding LSR, alternatively said cell has had the expression of endogenous LSR inhibited by transfection of a polynucleotide encoding a zinc finger protein of the invention, or by providing a chimeric oligonucleotide of the invention to the cell.

Other characteristics and advantages of the invention are described in the Brief Description of the Figures and the Examples. These are meant to be exemplary only, and not to limit the invention in any way.

BRIEF DESCRIPTION OF THE FIGURES Figures IA and IB show the effect of leptin on postprandial plasma TG response in db/db and dbPu/dbP' mice. Overnight-fasted db/db dbP"/dbP^ mice were gavage-fed a high-fat test meal and immediately injected intravenously (db/db) or intraperitoneally (db^/db^ with saline (open symbols) or 50 pg mouse recombinant leptin (closed symbols). At the indicated times, blood was collected from the tail or orbital vein, plasma was separated by centrifugation, and plasma TG concentrations were determined using an enzymatic kit. Each point represents the mean SEM (db/db: saline, n 4, leptin, n 3; dbP'/db^ saline, n 6, leptin, n The average plasma lipid response in 10 control C57BL6 mice is shown as WO 01/21647 PCT/IB00/01470 59 a dotted line in both A and B. In a separate experiment, shown as an inset for each strain of mice, overnight fasted db/db or db'" mice were gavage-fed the test meal and immediately injected intravenously with increasing concentrations of leptin. The plasma lipid response was then measured as in A and B. The area under the response curve (AUC) was then calculated using a triangulation method on Microsoft Excel between 0 and 4 hr (mg TG h mL). Values are presented as of control value (test meal alone obtained in A or Each point represents the mean of at least 3 mice.

Figure 2 shows 2 SI-Leptin binding to partially purified rat LSR. Aliquots (72 jg) of partially purified rat liver LSR were separated on a 4%-12% SDS-gradient polyacrylamide gel, and transferred to nitrocellulose as described previously (Yen Masson Clossais-Besnard

N.,

Andre Grosset Bougueleret Dumas Guerassimenko, and Bihain B.E.

(1999). J Biol Chem 274, 13390-13398) a gel strip stained with Coomassie blue is shown in lane 1. The nitrocellulose strips were washed, blocked, and incubated with anti-rat LSR protein antiserum (1:1000 dilution)(lane or with 200 ng/mL 2 SI-leptin (lane The strips were washed and bands were detected as described herein. Image analysis of lane 3 was performed on a Phosphorimager (Molecular Dynamics).

Figures 3A, 3B, 3C, and 3D show the effect of LSR subunit transfection on leptin binding and degradation in CHO-KI cells. For Fig. 3A, CHO-KI cells were transfected with increasing concentrations of the a, or P LSR plasmid, or vector alone using Fugene transfection reagent. After 48 h, the cells were washed once in PBS and incubated at 37 °C for 2 h with 10 ng/mL of'" 5 1-leptin in DMEM containing 0.2% BSA, 2 mM CaCI 2 and mM HEPES, pH 7.4 (Buffer The monolayers were washed and lysed with 0.1 N NaOH containing 0.24 mM EDTA, and the lysates were counted. The results are shown as the amount of cell-associated '"I-leptin. For Fig. 3B, lysates were prepared from CHO-KI wild type, stable transfectants of vector or LSR a' subunit, and PLC, and separated on a 10% SDSpolyacrylamide gel under denaturing and reduced conditions. After transfer to nitrocellulose, Western blots were performed using anti-LSR 170 antibody (can also be done with the human equivalent, 93A). Northern blots were done to detect LSR mRNA in CHO-KI wild-type versus PLC. RT-PCR analysis was also done in CHO-K 1 as compared to PLC. For Fig. 3C and 3D, confluent monolayers of stable-transfected cell lines expressing LSR a' subunit or vector alone were washed once in PBS and incubated at 37 OC for 2 h with increasing concentrations of '"I-leptin in Buffer A. The amount of cell-associated (Fig. 3C) and degraded (Fig. 3D) '"I-leptin was then measured as described herein. Results are shown as the mean of triplicate determinations.

WO 01/21647 PCT/IB00/01470 Figures 4A, 4B, 4C, and 4D show LSR binding and degradation of '"I-leptin in human hepatocytes, and the effect of 81B anti-LSR antibody. For Fig.4A, PLC cells were lysed (3-TI75 cm 2 flasks per condition) and immunoprecipitated with irrelevant or 81B anti-serum. The immunoprecipitates were washed, were separated on 10% SDS-polyacrylamide gels under nondenaturing conditions, and were transferred to nitrocellulose. Ligand blots using 25 I-leptin were then performed as described in Figure 2. For Fig. 4B, confluent monolayers of PLC cells were incubated at 37 °C for 30 min with 100 nM insulin, were washed, and then were incubated for 30 min at room temperature in the presence of anti-LSR peptide 81B antibody or irrelevant IgG. After this, the cells were incubated at 37 OC for 2 h with increasing concentrations of 2 I-leptin in Buffer A. The monolayers were washed, and the amount of 25 1leptin degraded was determined as described herein. Results are shown as the mean of duplicate (irrelevant IgG) or triplicate (anti-LSR peptide IgG) determinations. Fig. 4C is a schematic diagram of the motifs found from the predicted protein sequence of LSR a cDNA. A corresponding Kyte-Doolittle hydrophilicity plot (Lasergene, DNAstar, Madison, WI) is shown underneath. For Fig. 4D, PLC cell aliquots were prepared and incubated with irrelevant, 93A or 81B antibodies. After washing and incubation with goat-anti-rabbit FITC-conjugated antibody, the cells were fixed and analyzed by flow cytometry using a FACSCalibur (Becton Dickinson).

Figures 5A, 5B, 5C, and 5D show the stimulatory effect of leptin on LSR activity in PLC and suppression of this effect by 81B antibody. For Fig. 5A, 5B, and 5C, cultured PLC cells were incubated at 37 oC for 30 min with increasing concentrations of human recombinant leptin in Buffer A. After this, 0.5 mM oleate and 20 pg/mL 1'"I-LDL were added, and cells were further incubated at 37 °C for 2 h. Cells were washed, and the amount ofoleate-induced '2I- LDL bound internalized (5B) and degraded (5C) were measured. For Fig. 5D, PLC cells were incubated at room temperature for 30 min with 200 pg/mL anti-LSR peptide 81B or 170 antibody, followed by incubation at 37 OC for 30 min without (open bar) or with (hatched bar) ng/mL human leptin. Oleate (0.5 mM) and 25 1-LDL (20 pg/mL) were added, and the monolayers were left at 37 OC for 3 h. After washing, the amount of '2I-LDL binding was determined, and is shown here as the mean SD of triplicate determinations.

Figures 6A, 6B, and 6C show the effect of leptin on 1 2 sI-LDL and '"I-chylomicron binding to LSR in primary cultures of rat hepatocytes. For Fig. 6A, primary cultures of rat hepatocytes (48 hours after plating) were incubated at 37 OC for 30 min in the absence or presence of 50 ng/mL leptin in Buffer A, followed by a 20 min incubation at 37 OC with mM oleate. The cells were then washed with ice-cold PBS, precooled for 10 min, and then incubated for 1 h at 4 oC with increasing concentrations of 25 I-LDL in Buffer A. Cells were washed, were lysed in 0.1 N NaOH and were counted for radioactivity. Results are shown as the mean of duplicate determinations. For Fig. 6B, primary cultures of rat hepatocytes were WO 01/21647 PCT/IB00/01470 61 incubated at 37 °C for 30 min with or without 20 ng/mL leptin followed by incubation at 37 °C for 4 h with 6 pg protein/mL 2 "I-chylomicrons in the absence or presence of 0.5 mM oleate in Buffer A. The cells were then washed and the "'I-chylomicrons bound to the cell surface were released into the media by incubation with 10 mM suramin. The media was recovered and the radioactivity was measured. Results are shown as the mean SD of six determinations. Fir Figure 6C, after incubation at 37 OC for 30 min with 50 ng/mL leptin, the cells were incubated at room temperature for 30 min with 200 pg lgG/mL antibodies directed against rat LSR protein or irrelevant IgG. The amount of '"I-chylomicrons bound was determined, and results are shown as means SD of triplicate (irrelevant) or quadruplicate (anti-LSR) determinations.

Figures 7A and 7B show a comparison of the effect of human and mouse leptin on LSR activity in rat hepatocytes and on postprandial increase in plasma TG in db p mice. For Fig. 7A, primary cultures of rat hepatocytes were incubated 30 min at 37 °C without (open bar) or with 10 ng/mL recombinant human (solid bar) or mouse (hatched bar) leptin in Buffer A.

Oleate (0.5 mM) and 2 SI-LDL (20 pg/mL) were added and the cells were incubated 2 h at 37 OC.

The media were removed and were analyzed for TCA-soluble degradation products. The mean of duplicate determinations is shown. For Fig. 7B, db^" /db p mice were given a test meal as previously described, followed immediately by injection i.p. of saline (open bar, n human leptin (I pg/animal; solid bar, n 3) or mouse leptin (0.25 pg/animal; hatched bar; n The data represent the difference in TG concentrations measured at t=0 and the average of the concentrations at 3 and 4 hours. Results are shown as mean SEM.

Figures 8A and 8B show the effect of mouse or human leptin on LSR activity in primary cultures of rat hepatocytes or a human liver cell line (PLC). Primary cultured rat hepatocytes were obtained commercially (In Vitro Tech). The PLC cell line was obtained from ATCC repository and maintained in culture. Rat hepatocytes 72 h after plating (8A) or confluent monolayers of PLC (8B) were incubated 30 min at 37 oC with 0 (closed bar) or 10 ng/mL of human (open bar) or mouse (hatched bar) recombinant leptin. Following this, 0.5 mM oleate and 20 pg/mL LDL were added and the cells were further incubated for 2 h at 37 The cells were washed, and the amount of oleate-induced '2'I-LDL bound, internalized and degraded was measured as described previously (Bihain, and Yen, F.T. (1992). Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 31, 4628-4636.. Results here are shown as of control values obtained in the absence of leptin. Figure 9 shows the effect of mouse or human leptin peptide on LSR activity in PLC. Confluent PLC monolayers were incubated 30 min at 37 °C with increasing concentrations of mouse (U or human leptin peptide. Following this, 0.5 mM oleate and 20 pg/mL 2 I-LDL were added and the cells were further incubated for 2 h at 37 The cells were washed, and the amount of oleate-induced WO 01/21647 PCT/IB00/01470 62 2 "I-LDL bound and degraded was measured as described previously (Bihain, and Yen, F.T.

(1992). Free fatty acids activate a high-affinity saturable pathway for degradation of low density lipoproteins infibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 31, 4628-4636.

Figure 10 shows the effect of mouse or human leptin peptide on LSR activity in primary cultured rat hepatocytes. Cells were incubated 30 min at 37 OC with increasing concentrations of mouse (U or human leptin peptide. Following this, 0.5 mM oleate and 20 pg/mL '"I-LDL were added and the cells were further incubated for 2 h at 37 oC. The cells were washed, and the amount of oleate-induced '5I-LDL bound and degraded was measured as described previously (Bihain, and Yen, F.T. (1992). Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 31, 4628-4636).

Figures 11A and 1 IB show the effect of mouse leptin (Fig. 1 1A) or leptin peptide (Fig.

11 B) on postprandial plasma TG response in ob/ob mice. A single dose of 50 ng mouse leptin M leptin peptide or a comparable volume of saline was injected subcutaneously at t 0 h (8:30 AM) directly following gavage of a high fat meal (0.5 mL).

Postprandial triglyceridemia was measured as described previously. Each point represents the mean SEM saline, n 8, leptin, n 7; leptin peptide: saline, n 8, leptin, n The insert to figure 11B shows the effect of mouse leptin on LSR activity in primary cultures of rat hepatocytes. Primary cultures of rat hepatocytes were incubated 30 min at 37 OC with increasing concentrations of mouse leptin peptide. Oleate (0.5 mM) and 1 2 SI-LDL (20 pg/mL) were added and the cells were incubated 2 h at 37 After washing, the amount of 1251-LDL bound to the cell surface was measured as described in the Examples section herein.

Figure 12 contains a Table that presents results showing the effect of test meal with and without leptin injection on postheparin lipolytic activity in mice. Animals were gavaged with the test-meal and injected at the same time with 50 Pg leptin or physiological saline as described previously (t 0 After I h, the mice were injected with heparin and blood samples were taken at the peak of postprandial triglyceridemia (t 2 Lipase activity was measured in the postheparin plasma as described in the Examples section herein,, and is reported here as the mean SEM (n 3 animals for each condition; ns not significant).

Figure 13 shows a multiple sequence alignment of leptin polypeptides from various species including: Homo sapiens (SEQ ID NO:32), Mus musculus (SEQ ID NO:34), Rattus norvegicus (SEQ ID NO:38), Sus scrofa (SEQ ID NO:39), Bos Taurus (SEQ ID NO:28), Gallus gallus (SEQ ID NO:30). Ovus aries (SEQ ID NO:35), Canisfamiliaris (SEQ ID NO:29), Gorilla gorilla gorilla (SEQ ID NO:31), Macaca mulatta (SEQ ID NO:33), Pan troglodytes (SEQ ID WO 01/21647 PCT/IB00/01470 63 NO:36), and Pongo pygmaeus (SEQ ID NO:37). Divergent residues (from the consensus sequence) are boxed. The 22 amino acid region of the exemplary active leptin peptide is shaded for all species in the alignment. Residues 10-13 of the shaded region make up the "leptin fragment central sequence".

Figure 14 shows a schematic diagram of an exemplary retroviral vector. The vector pMX-IRES-GFP contains the murine Moloney virus LTR and a packaging signal (Onishi et al.

Exp. Hematol. 24: 324-329 (1996)); the EMCV IRES is placed between the polylinker/stuffer and a cDNA encoding a selectable marker protein. Three exemplary marker proteins are GFP, murine CD2 and human CD4. The IRES sequence is indicated as a shaded box with an arrow indicating the direction of translation. The segment containing the bacterial origin of replication and ampicillin resistant gene is indicated by a black box. The stippled box represents sequence encoding the green fluorescent protein; alternatively it can contain the truncated CD2 or CD4 sequences. Open boxes with arrows indicate the viral LTR sequences. The open box indicates a stuffer fragment containing multiple cloning sites.

Figure 15 shows a schematic of a plan to create truncated LSR constructs.

Figures 16A, 16B, and 16C show that the transfection of a truncated form of LSR 6) increases 12 5I-LDL binding uptake and degradation in PLC cells in reference to protein concentration. All points are done in triplicate.

Figures 17A, 17B, and 17C show that the transfection of a truncated form of LSR (DN5+6) increases 25 I-LDL binding uptake and degradation in PLC cells correcting for transfection efficiency using P-gal as a reference. All points are done in triplicate.

Figures 18A and 18B show graphs of the total LSR expression in mouse liver determined by Quantitative PCR.

Figures 19A and 19B show graphs of the expression of LSR isotypes in mouse liver.

Figures 20A and 20 B show graphs of the relative levels of LSR isotype expression in mouse liver.

Figure 21 shows a graph of total LSR expression in mouse brain determined by Quantitative PCR.

Figure 22 shows a graph of the expression of LSR isotypes in mouse brain.

Figure 23 shows a graph of the relative levels of LSR isotype expression in mouse brain.

Figures 24A, 24B, 24C, 24D, and 24E show the difference in LSR expression and activity in 2 cultured human hepatocyte cell lines. Figures 24A and 24B show graphs of LSR mRNA levels (24A) and cell surface expression (24B) in PLC (GG) and HepG2 (AG) cells by quantitative PCR and FACS, respectively. Figures 24C, 24D, and 24E show graphs of the oleate-induced '"I-LDL bound internalized and degraded in confluent monolayers of PLC and HepG2 that were incubated 3 h at 37 OC with the indicated concentrations of WO 01/21647 PCT/IB00/01470 64 oleate and 20 ug/mL '"I-LDL. The cells were then washed and the amounts of '"I-LDL bound, internalized and degraded were measured as described previously.

Figure 25 shows a table of the characteristics of recombinant ZFPs directed toward LSR sequences. The first column is the identification number of the plasmid expressing a specifically engineered ZFP. The ZFP column represents different zinc finger "cassettes" designed to recognize the 9 bp regions of the target sequence. These "cassettes" have then been linked together (see WO 98/54311) to create the ZFP for the final 18 bp target sequence listed in the final column. Sangamo determined the data on the fold activation and binding constant. The target sequences are located 5' to the translation start site in the mouse LSR gene sequence.

Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 261, 26J, 26K, 26L, 26M, 26N, 260, 26P, 26Q, 26R, 26S, and 26T show schematics and nucleotide sequence of the LSR zinc finger plasmids pSBS5182-NVF (26A), pSBS5183-NVF (26B), pSBS5185-NVF (26C), pSBS5186- NVF (26D), and pSBS5205-NVF (26E). The locations of the ampicillin gene (Amp), neomycin gene (Neo) CMV promoter NLS, ZFP, VP 16, FLAG, bGHpA as well as various restriction sites are shown in the schematics.

Figure 27 shows a Northern Analysis of LSR zinc finger mRNA expression. Numbers are shown as percent of control plasmid. Only the results from 48 hrs are shown.

Figure 28 shows a more detailed Northern analysis of LSR zinc finger mRNA expression. Numbers are shown as percent of control plasmid. Only the results from 48hrs are shown.

Figure 29 shows a quantitative PCR Analysis of Hepal-6 cells transfected with ZFP- NVF constructs.

Figures 30A, 30B, 30C, 30D, 30E, and 30F show binding, uptake and degradation (BUD) data from ZFPs. The following ZFP's were examined: 5185-NVF, 5186-NVF, and control plasmid VegF-NVF (a non related ZFP). Results are corrected for total protein in A-C and for P-gal in D-F.

Figure 31 shows a diagram of the coculture system. Endothelial cells are plated in the upper compartment on the filter and astrocytes in the lower compartment on the plastic of the Petri dish.

Figure 32 shows a diagram of transcytosis and permeability studies.

Figure 33 shows a graph of leptin transcytosis in BBB in vitro model. Cells were incubated with '"I-leptin alone (10,000 dpm/ngXclosed squares), with 1 pg/mL unlabelled leptin (triangles), 50 pg/mL MP (circles), 50 pg/mL HP (open squares), or 2 mg/mL lactoferrin (asterisks).

Figure 34A and 34B show graphs of the effect of leptin, MP, HP, and lactoferrin on the permeability of the EC monolayer. Sucrose (34A) and inulin (34B) permeability studies were WO 01/21647 PCT/IB00/01470 performed in the absence (diamonds) or presence of 10 ng/mL leptin (squares), 5 pg/mL leptin (triangles), 10 pg/mL leptin (crosses). The effect of peptides were also tested by the addition of ng/mL leptin 50 ug/mL mouse peptide (MP, circles) or 10 ng/mL leptin+50 pg/mL human peptidc (HP, open squares) or 10 ng/mL leptin+2 mg/mL lactoferrin (lacto, asterisks).

Figures 35A and 35B show graphs of LSR activity and mRNA expression measured in PLC cells preincubated 24 h with leptin. In Figure 35A, PLC monolayers were incubated 24 h at 37 °C with or without 200 ng/mL human recombinant leptin. After washing with PBS, cells were incubated 30 min at 37 °C with increasing concentrations of human leptin, followed by a 2 h incubation at 37 OC with 0.8 mM oleate and 20 pg/mL 2 sI-LDL. Cells were washed, and the amount of oleate-induced '"I-LDL binding was measured as described previously.

Results are shown as the mean of triplicate determinations. In Figure 35B, PLC monolayers were incubated 24 h at 37 OC with 0, 200, or 400 ng/mL human recombinant leptin. After washing with PBS, the cells were harvested. Total RNA was prepared from the cell pellets, and Northerns were performed to detect LSR mRNA, using GAPDH probe as loading control as described previously. Northern blots were scanned on the Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Densitometric analysis of the images was performed using the software ImageQuant. Results are shown as the amount of LSR signal relative to that of GAPDH (mean SD, n 3/condition).

EXAMPLES

The following Examples are provided for illustrative purposes and not as a means of limitation. One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein all of which form part of the instant invention.

GENERAL MATERIALS and METHODS Materials 125 Na I was purchased from Amersham-Pharmacia (Piscataway, NJ; Les Ulis, France).

Oleic acid, bovine serum albumin (A2153)(BSA), were obtained from Sigma (St. Louis, MO; St.

Quentin Fallavier, France). Sodium heparin was purchased from Choay laboratories (Gentilly, France). Fugcne was purchased from Roche Boehringer Mannheim (Indianapolis, IN), and Superfect from Qiagen (Valencia, CA). Zeocin was obtained from Invitrogen (Carlsbad, CA).

Suramin was a gift from Bayer Pharmaceuticals (Puteaux, France). Enzymatic kits for the determination of TG and FFA were obtained from Roche-Boehringer Mannheim (Meylan, France) and WAKO (Richmond, VA; Unipath, Dardilly, France), respectively. Dulbecco's modified Eagle's medium (DMEM), trypsin, penicillin-streptomycin, glutamine, and fetal bovine WO 01/21647 PCT/IB00/01470 66 serum (FBS) were purchased from Life Technologies, Inc (Grand Island, NY; Eragny, France).

RIA kits for plasma leptin measurements were obtained from Linco (St. Louis, MO).

Experiments in Figures 1 (db/db only), 2 and 6 were performed using recombinant mouse leptin prepared in the laboratory as described previously (Yen Masson Clossais-Besnard N., Andre Grosset Bougueleret Dumas Guerassimenko, and Bihain B.E.

(1999). JBiol Chem 274, 13390-13398).

The remainder of the experiments were performed using commercial preparations of recombinant human or mouse leptin (Sigma and Calbiochem, Meudon, France). a 2 Macroglobulin-methylamine was a kind gift from Dr. D. Strickland (American Red Cross, Rockville, MD).

Animals Male wild-type and C57BL/Ks db/db (db) mice were purchased from R. Janvier Breeding Center (Le Genest St. Isle, France), while male db"'/dbPa" were kindly made available by Prof. J.L. Guenet (Institut Pasteur, Paris, France). Female ob/ob mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). All animals were housed in an animal facility on a 12 h light/dark cycle and were allowed water and rodent chow (No. 113, UAR, Epinay-sur- Orge, France) ad libitum. Mean body weights at the time of the experiment for wild-type, db/db, db"/db p and ob/ob mice were 27.8 1.4, 33.8 9, 74.6 11.4 g, and 49.4 5.49 g, respectively. The research protocol was in accordance with French Ministry of Agriculture, section of Health and Animal Protection and the established institutional guidelines.

Cells Primary cultures of rat hepatocytes were prepared as described previously (Yen, F.T., Mann, Guermani, Hannouche, Hubert, Homick, Bordeau, V.N., Agnani, and Bihain, B.E. (1994). Biochemistry 33, 1172-1180). using overnight-fasted 150- 200 g Sprague-Dawley male rats Janvier Breeding Center) or obtained commercially (InVitro Technologies, Baltimore, MD). Cells were used in experiments 48 h after plating. The PLC liver hepatoma (CRL-8024) and Chinese hamster ovary (CHO-K 1, CRL 9618) cell lines were obtained from the ATCC repository (CRL-8024; Manasass, VA). The PLC line was maintained in tissue culture with MEM containing 10% FBS, 2 mM glutamine, sodium pyruvate, nonessential amino acids, 100 units/mL penicillin, and 100 units/mL streptomycin. CHO-K1 cells were grown in Ham's-F12 containing 10% FBS, 2 mM glutamine and 100 units/mL each of penicillin and streptomycin.

WO 01/21647 PCT/IB00/01470 67 Anti-LSR antibodies and peptides The preparation of antibodies directed against rat LSR protein, and anti-LSR peptide 170 antibodies was as described previously (Yen Masson Clossais-Besnard Andre P., Grosset Bougueleret Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398. Synthetic peptides 81B and 93A with sequences corresponding to human LSR ac residues 35-45 (FGRDARARRAQ) and 613-627 (EEAYYPPAPPPYSET), respectively, were obtained commercially. Polyclonal antibodies directed against this synthetic peptide conjugated to KLH were prepared, and the IgG fraction was purified as described previously (Yen Masson Clossais-Besnard Andre Grosset Bougueleret L.

Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398.) Synthetic peptides corresponding to residues 117-138 of mouse leptin (CSLPQTSGLQKPESLDGVLEAS) as well as the corresponding fragment of human leptin were commercially prepared (Research Genetics, Huntsville, AL).

In Vivo Methods Measurement of plasma lipid response in mice Mice that were fasted for 2-3 hours were gavage-fed 300 tL of a test meal consisting of fat (37% saturated, 27% mono-, and 36% polyunsaturated fatty acids), 20% protein and carbohydrate, and providing 56 kcal ofenergy/kg (1.5 g butter, 1.5 g sunflower oil, 2.5 g nonfat dry milk, 2.5 g sucrose and 3 ml water). Immediately after the meal, the animals were injected intravenously (db/db) or intraperitoneally (dbP"/dbP") with either 200 plL physiological saline or 200 L of the same solution containing recombinant mouse leptin. At selected time intervals, 20 pL of blood were collected from the orbital (dbP"'/db' or tail (db/db) vein into ice-cold microfuge tubes containing 4 mmol/L EDTA. Plasma was obtained by centrifugation at 2500 rpm for 20 min at 4 and was frozen as aliquots at -80 *C before analysis. TG concentrations were determined using a commercially available enzymatic kit with controls included in each assay (Precinorm L, Roche-Boehringer Mannheim; Lyotrol N, BioMerieux).

Measurement of postheparin lipolytic activity Mice were gavage-fed and injected with leptin or control solutions as described above.

At t 1 h, the mice were injected subcutaneously with heparin (100 IU/kg body weight). At t 2 h, the animals were bled and the plasma was immediately separated by centrifugation. Lipase activity was determined according to Iverius and Brunzell (1985) using 20% Lipoven (Fresenius France Pharma, Louviers, France) as the source of TG. The assay was performed using 25 pL postheparin plasma in 0.15 M NaCI (200 pL total volume), and in the presence of 10 pL heat- WO 01/21647 PCT/IB00/01470 68 inactivated (56 OC, 30 min) human plasma as a source of apoC's. Before and at the end of the incubation, FFA concentrations were determined using an enzymatic kit.

Cell Culture Studies Lipoprotein receptor studies LSR activity was measured as the oleate-induced binding, uptake, and degradation of I-low density lipoprotein (LDL) in cells following the method described in detail previously and Yen, F.T. (1992). Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 31,4628-4636; Yen, Mann, Guermani, L.M.

Hannouche, Hubert, Horick, Bordeau, Agnani, and Bihain, B.E (1994). Biochemistry 33, 1172-1180); Yen Masson Clossais-Besnard Andre P., Grosset Bouguclcret Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398). Modifications of the standard protocols are described in the Brief Description of the Drawings.

Identification Of LSR Protein Western blotting Confluent monolayers of cells were washed in PBS, and lysed in 20 mM Tris containing 2 mM EDTA and 0.5 SDS and an protease inhibitors (0.1 mg/mL PMSF, 2 pg/mL leupeptin and 1.9 ug/mL aprotinin). The lysate was then separated on 10% SDS-PAGE under denaturing conditions. After transfer to nitrocellulose, the strips were probed with anti-LSR peptide anti-serum. Bands were revealed after incubations with secondary goat anti-rabbit IgG conjugated to alkaline phosphatase. After washing in PBS containing 0.5% Tween 20, the bands were revealed by incubation with substrate.

Immunoprecipitation Confluent monolayers of PLC cells were lysed in PBS containing 1% Triton X-100, and then were incubated with the specified anti-LSR antibodies, as described previously (Yen F.T., Masson Clossais-Besnard Andre Grosset Bougueleret Dumas J.B., Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398).

Immunoprecipitates were separated on 10% SDS-polyacrylamide gels under nondenaturing conditions, and then transferred onto nitrocellulose.

Ligand blotting Partially purified rat LSR (240 kDa band complex) was obtained as described previously WO 01/21647 PCT/IB00/01470 69 (Yen Masson Clossais-Besnard Andre Grosset Bougueleret Yen F.T., Masson Clossais-Besnard Andre Grosset Bougueleret Dumas J.B., Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398.) The band was separated on non-denaturing 4-12% gradient SDS polyacrylamide gel, and was transferred to nitrocellulose by semi-dry transfer (Biorad, 18 V, 25 min). The nitrocellulose strip was incubated at room temperature with PBS containing 3% BSA, and then incubated at 37 *C for I h with 200 ng/mL 125I-leptin in PBS containing 0.2% BSA, pH 7.4. After six 10 min washes in PBS containing 0.5% TritonX-100, the strip was air-dried and exposed on a phospohor screen for analysis.

Preparation of lipoproteins Human LDL (1.025 d 1.055 g/mL) were isolated by sequential ultracentrifugation of fresh plasma obtained from the local blood bank (Havel, and Kane, J.P. (1995). In The Metabolic and Molecular Basis of Inherited Disease, vol. II, Scriver, Beaudet, A.L., Sly, and Valle, eds. (New York, NY: McGraw-Hill, Inc), pp. 1841-1851.

Rat chylomicrons were prepared from overnight-fasted male Sprague-Dawley rats (300- 400 g) given a high-fat liquid meal similar to that given to mice (2 mL per animal). After min, the animals were anesthetized and catheters were inserted in the main abdominal lymph duct. Lymph was collected over 2 hours, and the chylomicrons were isolated. Contaminating albumin was removed by incubation for 30 min at room temperature with an equivalent volume of swollen Blue Sepharose CL-6B gel (Amersham Pharmacia Biotech) (Mann, Troussard, Yen, Hannouche, Najib, Fruchart, Lotteau, Andre, and Bihain, B.E. (1997). J. Biol. Chem. 272, 31348-31354). All lipoproteins were stored in the dark at 4 °C under N 2 and used within 2 weeks (LDL) or 3 days (chylomicrons) of their isolation Radiolabelling Lipoproteins were radioiodinated using Bilheimer's modification of the McFarlane's procedure (Bilheimer, et al. (1972). Biochim. Biophys. Acta 260, 212-221), and used no more than 1 week after radiolabeling. 125 1-LDL was filtered (0.2 pm, Gelman, Ann Arbor, MI) on the day of the experiment.

Leptin was iodinated using lodobeads (Pierce) according to the manufacturer's instructions.

WO 01/21647 PCT/IB00/01470 Cloning Of Full Length cDNA Human LSR Human homologous sequences of rat LSR cDNA were found with 2 partially overlapping human genomic sequences (Genbank accession nos: AD000684 and AC002128).

ESTs generated on the basis of these sequences were used to screen a human BAC library. A single clone was isolated and sequenced. Analysis of this sequence revealed several variations from the public sequence. A revised LSR sequence is currently available in Genbank (accession numbers TBA).

An 805 bp fragment was obtained by PCR amplification of human liver mRNA (Sense primer: CTACAACCCCTACGTCGAGT (SEQ ID NO:22), antisense primer: 5' AGGCGGAGATCGCCAGTCGT (SEQ ID NO:23)), and subcloned into the TA cloning vector (Invitrogen, Carlsbad, CA). The cloned insert was isolated by digestion with EcoRl, was purified (GenClean kit, Bio 101, Vista, CA), and the DNA was labeled with a- 3 P-dCTP (NEN, Boston, MA) using the random primers labeling system (Life Technologies). The labelled fragment was used to screen the cDNA library (Superscript, Life Technologies), from which we obtained a partial a' clone (clone 18251), lacking 161 bp of the 5' region.

The missing 5' region was obtained by PCR amplification (AmpliTaq, Promega, Madison, WI) from a first strand cDNA prepared from human liver total RNA (Clonetech, Palo Alto, CA)(both oligo dT and random primers were used). The primers for PCR were sense 5'CCTTTGTCCACGTCGTTTACGCTC-3' (SEQ ID NO:24) and antisense TCACAGCGTTGCCCTGCTTG (SEQ ID NO:25). The PCR was performed with annealing temperature of 65 *C and 35 cycles. The fragment was cloned into pGEMT-Easy Vector (Promega).

Fragments corresponding to the a forms and 0 were cloned into pGEMT-Easy Vector and then used to replace the appropriate region in the LSR a' clone. The full-length LSR a, a', and P clones were reconstructed in pTracer-CMV2 vector (Invitrogen) using EcoRI/Xba I.

PCR Analysis of Human LSR Similarly to previous results with rat LSR (Yen Masson Clossais-Besnard Andre P., Grosset Bougueleret Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398), two splice variants of LSR were detected by RT-PCR analysis of human hepatocyte cDNA. In Fig. 3B, sense and antisense primers were designed to yield three products, of which two were the splice variants. The primer sequences were: sense, TTACTGCTCCGTGGTCTCAGC- 3' (SEQ ID NO:26) and antisense, AGCTACTCCTGTCAACGTCTCC 3' (SEQ ID NO:27). Identities of each band were confirmed by sequencing.

WO 01/21647 PCT/IB00/01470 71 Northern Blotting Northern blots were performed as described previously using as a probe clone 18251 described above (Yen Masson Clossais-Besnard Andre Grosset Bougueleret L., Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398).

In vitro translation In vitro translation products were obtained using "S-methionine (Amersham) and the T7 coupled transcription/translation kit from Promega.

Transient Transfection Studies CHO-K cells were plated at a density of 300,000 cells/36 mm dish the day before transfection. After 24 h, plasmid preincubated with Fugene transfection reagent was added to the cells, which were further incubated at 37 OC. Cells were used 48 h after transfection as described in the Brief Descriptioon of the Figures.

Stable Transfections Stable transfectants were prepared from CHO-KI cells using Superfect according to the manufacturer's instructions. After introduction of the plasmid into the cell with Superfect, the cells were grown in the presence of 750 pg/mL zeocin. After elimination of untransfected cells, the antibiotic concentration was reduced to 500 pg/mL. Clones were isolated using cloning cylinders, and maintained in tissue culture media containing 100 pg/mL zeocin.

FACS Analysis Flow cytometry is a laser-based technology that is used to measure characteristics of biological particles. The underlying principle of flow cytometry is that light is scattered and fluorescence is emitted as light from the excitation source strikes the moving particles.

Assay 1 :PLC cell suspensions were obtained using non-enzymatic dissociation solution (Sigma), and then were incubated for I h at 4 OC with a 1:200 dilution of anti-LSR 81 B or irrelevant anti-serum in PBS containing 1% BSA. After washing twice with the same buffer, goat anti-rabbit FITC-conjugated antibody (Rockland, Gilbertsville, PA) was added to the cells, followed by a further incubation for 30 min at 4 OC. After washing, the cells were fixed in 2% formalin. Flow cytometry analysis was done on a FACSCalibur cytometer (Becton- Dickinson, Franklin Lakes, NJ).

WO 01/21647 PCT/IB00/01470 72 Assay 2 Cells are cultured in a T175 flasks according to manufacturer's instructions for 48 hours prior to analysis.

Cells are washed once with FACs buffer (lx PBS/2% FBS, filter sterilized), and manually scraped from the flask in 10 mLs of FACs buffer. The cell suspension is transferred to a 15 mL conical tube and centrifuged at 1200 rpm, 4 OC for 5 minutes. Supernatant is discarded and cells are resuspended in 10 mL FACs buffer chilled to 4 A cell count is performed and the cell density adjusted with FACs buffer to a concentration of 1 xl0 6 cells/ mL. One milliliter of cell suspension was added to each well of a 48 well plate for analysis. Cells are centrifuged at 1200 rpm for 5 minutes at 4 Plates are checked to ensure that cells are pelleted, the supernatant is removed and cells resuspended by running plate over a vortex mixer. One milliliter of FACs buffer is added to each well, followed by centrifugation at 1200 rpm for minutes at 4 This described cell washing was performed a total of 3 times.

Primary antibody, titered in screening experiments to determine proper working dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells in a total volume of 50 pL FACs buffer. Plates are incubated for h at 4 °C protected from light. Following incubation, cells are washed 3 times as directed above.

Appropriate secondary antibody, titered in screening experiments to determine proper working dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells in a total volume of 50 L FACs buffer. Plates are incubated for Ih at 4 °C protected from light. Following incubation, cells are washed 3 times as directed above. Upon final wash, cells are resuspended in 500 uL FACs buffer and transfered to a FACs acquisition tube. Samples are placed on ice protected from light and analyzde within 1 hour.

Protein Determinations Protein concentrations were determined using Markwell's modified Lowry procedure (1981) or BCA protein assay (Pierce Chemical Co, Rockford, IL) and BSA as standard.

Statistical Analysis Results were analyzed using unpaired Student's t-test.

EXAMPLE 1: Effect of Leptin on Postprandial Plasma TG Response Transient hypertriglyceridemia seen after administration of a test meal in two strains of obese mice with defects of the Ob-Receptor (OB-R) is shown in Figures 1A and 1B (open symbols). The db/db mice present a mutation of the Ob-Rb isoform, preventing signaling to the JAK and Stat system, while the dbP'/dbP^ lack any leptin signaling capacity through the Ob-R.

WO 01/21647 PCT/IB00/01470 73 Similar to what is observed in most obese human subjects (Lewis, O'Meara, Soltys, Blackman, Iverius, Druetzler, Getz, and Polonsky, K.S. (1990) J.

Clin. Endocrinol. Metab. 71, 1041-105o; Vansant, Mertens, and Muls, E. (1999) Intl. J.

Obesity 23, 14-21) postprandial plasma lipid levels were elevated in both strains of obese mice when compared to lean controls (shown as dotted lines). A single bolus injection of 50 uLg leptin at the time of the meal decreased the amplitude of the triglyceride response (Fig. 1A and 1B, closed symbols); this effect could not be attributed to a reduction in food intake since the meal was administered by intragastric cannulation.

A significant reduction of the area under the TG curve was observed with 250 ng of leptin per animal (Fig. IA, IB, insets). It can be estimated (average body weight ofdbP"/db 74.6 11.4 g; plasma volume 45 mL per kg) that this dose cannot cause more than a two-fold increase of the concentration of circulating leptin (86.7 12.2 ng/mL) in db"'/db

P

Maximum effect of leptin was achieved with 500 ng per animal which decreased by 80% and 65% the area under the postprandial TG curve in db/db and respectively. This dose of leptin (7 ug per kg body weight) is 15-fold lower than that used to achieve 30 to 40% reduction of food intake after peripheral administration of leptin (Campfield, Smith, Guisez, Devos, and Bum, P. (1995) Science 269, 546-549; Halaas et al, 1995; Halaas, Gajiwala, K.S., Maffei, Cohen, Chait, Rabinowitz, Lallone, Burley, and, J.M.

(1995) Science 269, 543-546; Pelleymounter, Cullen, Baker, Hecht, R., Winters, Boone, and Collins, F. (1995) Science 269, 540-543). These data establish that leptin can control the exogenous lipoprotein pathway and that this regulation occurs in dbP'/db' in spite of the complete defect of the Ob-R.

EXAMPLE 2: Leptin Binding to Rat LSR The binding of leptin to LSR was tested using partially purified rat LSR multimeric complexes. Complexes separated by SDS electrophoresis (Fig. 2, lane 1) and transferred to nitrocellulose, bound 1 25 I-leptin (Fig. 2, lane The same bands were recognized by polyclonal anti-rat LSR antibodies (Fig. 2, lane The specificity of these antibodies has been described previously (Yen Masson Clossais-Besnard Andre Grosset Bougueleret L., Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398).

EXAMPLE 3 Effect of LSR Suhunit Transfection To determine which of the LSR subunits is responsible for leptin binding, CHO-K1 cells were transiently transfected with increasing concentrations of each of the 3 human LSR plasmids (Fig. 3A). CHO-KI cells were selected because they had the lowest level of endogenous LSR expression of the different cell lines tested. This level is far lower than that of a human WO 01/21647 PCT/IB00/01470 74 hepatocyte cell line (PLC) used to systematically characterize human LSR activity (Fig. 3B).

The data showed that only transfection with the LSR a' plasmid increased the binding of leptin to CHO-K1 cells (Fig. 3A). Leptin binding to CHO-KI cells transfected with LSR P or a remained at levels similar to those seen with the vector alone. Analysis of the expression of cotransfected green fluorescent protein (GFP) estimated transfection efficiency at 25% for all 3 transiently transfected plasmids.

CHO-K1 cells stably expressing LSR a' were also obtained and were determined to have an increased 25 I-leptin binding and uptake (Fig. 3C). The apparent molecular mass of human LSR a' in stable CHO-KI transfectant cells corresponded to that of the smallest LSR subunit 70 kDa) in PLC cells (Fig. 3B). Lineweaver-Burk transformation of leptin binding to CHO-K1 cells expressing LSR a' yielded an estimated Kd of 1.3 nM (Fig. 3C, inset), 2 fold that of the Ob-R (Kd 0.7 nM; Tartaglia et al, 1995). Leptin binding to LSR a' led to its internalization and proteolytic degradation consistent with this leptin binding reflecting a biologically relevant process (Fig. 3D).

Similar to what is observed in cells transfected with the Ob-Ra or Ob-Rb (Uotani, Bjerbaerk, Torne, and Flier, J.S. (1999). Diabetes 48, 279-286.) the amount of'"I-leptin degraded by CHO-K cells transfected with LSR a' represented only 16% of that bound and internalized by the cells. These rates of 2 I-leptin degradation are much lower than those observed with receptors mediating rapid endocytosis (Goldstein, Basu, Brown, M.S. (1983). 98, 241-260). For instance, after 2 h incubation, the amount of "I2-LDL degraded through LSR represents 4-5 times the amount bound to the cell surface (Bihain, and Yen, F.T. (1992). Although not intending to be limited by any particular theory, the simplest explanation is that LSR a' lacks the di-leucine routing signal known to trigger rapid lysosomal delivery. The LSR a contains such a signal, consistent with previous observations that the a subunit is a critical element allowing LSR to function as lipoprotein receptor (Yen Masson Clossais-Besnard Andre Grosset Bougueleret Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398).

Similar experiments are performed in the other stable cell lines expressing the subunits of LSR alone or in all combinations (see table, below). These cell lines are useful for screening small molecules or any potential agonist or antagonist for activity against either the leptin or triglyceride (or both) activity of LSR. In addition, they can be employed in receptor binding assays using FACS analysis or radiolabelled ligands to identify additional ligands of LSR.

WO 01/21647 PCT/IB00/01470 LSR stable-transfectant Cell Lines CHO LSR alpha CHO LSR alpha' CHO LSR beta CHO LSR alpha'/beta CHO LSR alpha/beta CHO LSR alpha/alpha' CHO LSR alpha/alpha'/beta EXAMPLE 4 Effect of 81B anti-LSR Antibody on LSR Binding and Degradation of Leptin To test whether in nontransfected cells leptin binds to LSR, PLC cell lysates were immunoprecipitated with an antibody directed against a synthetic peptide with a sequence identical to LSR residues 35-45 (81B). Ligand blotting showed that 25I-leptin binds directly to the multimeric complexes (apparent molecular masses of 200 and 230 kDa) precipitated by the 81B antibody (Fig. 4A). These complexes are of a size similar to that of rat LSR multimeric complexes (Yen Masson Clossais-Besnard Andre Grosset Bougueleret L., Dumas Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398).

Significant amounts of TCA-soluble chloroform-insoluble 2 S-leptin degradation products were found in the incubation media after 2 h incubation of PLC cells with increasing concentrations of 25 1-leptin (Fig. 4B, The dose response curve indicated that the process saturated for leptin concentrations 10 ng/mL (Fig. 4B). The amount of leptin degraded per mg of PLC cell protein is about half as much as that degraded by CHO-K 1 LSR a'stable transfectants (Fig. 3D).

Chloroquine (50 M) inhibited 25 I-leptin degradation by more than 60%, while increasing the amount of cell-associated 2 1-leptin (2-4 fold). This is consistent with 25 1-leptin degradation occurring in lysosomes after receptor-mediated endocytosis. The 81B antibody that immunoprecipitated LSR multimeric complexes had a profound inhibitory effect on leptin degradation in PLC cells (Fig. 4B, This effect was maximal with 10 ng/mL of leptin and 200 jg/mL of antibody and was partially competed off by increasing leptin concentrations at ng/mL. Because immunoprecipitation data revealed no interaction of the 81B antibody with the Ob-R or any other protein (Fig. 4A), the inhibitory effect of this antibody on leptin degradation indicates that in cells of liver origin, the LSR is quantitatively the primary mechanism for leptin degradation. FACS analysis confirmed that the 81B anti-LSR antibody binds to non- WO 01/21647 PCT/IB00/01470 76 permeabilized PLC cells (Fig. 4D). This indicates that the amino-terminal is exposed on the cell surface.

Leptin binding to LSR does not require the presence of FFA and is inhibited by the 81B antibody directed towards the LSR sequence located near the amino terminal end Immunoinhibition studies previously showed that the cluster of charged residues found at the carboxyl terminal end most likely represents the rat LSR lipoprotein binding site (Yen, F.T., Masson Clossais-Besnard Andre Grosset Bougueleret Dumas J.B., Guerassimenko, and Bihain B.E. (1999). J Biol Chem 274, 13390-13398). Accordingly, LSR was classified as a type I1 membrane receptor. FACS analysis using the 170 antibody, directed towards a synthetic peptide with a sequence corresponding to that of LSR's carboxyl terminal end, is consistent with this interpretation (Fig. 4D).

While not wishing to be limited by any theory, the observation that the 81 B antibody inhibits leptin binding to LSR and binds to intact PLC cells (FACS analysis, Fig. 4D), suggests that LSR amino terminal ends are also exposed on the external side of the plasma membrane.

LSR contains a typical 28 amino acid transmembrane spanning domain located between residues 259-286 (Fig. 4C). In addition, a cluster consisting of 3 stretches of hydrophobic amino acids is located towards the amino terminal end. Each of these hydrophobic clusters is too short to allow crossing of the plasma membrane, but since the three hydrophobic elements are in close proximity with only two short hydrophilic separating clusters, a transmembrane spanning region could be constituted. In this case, the two separating hydrophilic domains would be oriented inwardly to minimize interaction with the hydrophobic moieties of the phospholipid bilayers.

According to this model, LSR a and a' could cross the plasma membrane twice, with both carboxyl and amino terminal ends protruding extracellularly. LSR P would be limited to a single crossing of the membrane.

EXAMPLE Effect of Leptin on LSR Activity The effect of leptin on the activity of LSR with respect to its ability to bind, internalize and degrade lipoproteins was also studied. Leptin directly increased the oleate-induced LSR binding uptake and degradation of 2 I-LDL in a dose-dependent manner (Fig. 5A, 5B, 5C). The effect was observed at leptin concentrations 10 ng/mL.

The specificity of leptin's stimulatory effect upon LSR was further established by the observation that leptin at concentrations of up to 2 pg/mL had no detectable effect on the degradation of LDL by the LDL-receptor nor on that of activated a2-macroglobulin, the preferred LRP ligand.

The stimulatory effect of leptin on LSR activity as a lipoprotein receptor was suppressed by the 81 B antibody (Fig. 5D). The antibody 170 directed against a rat LSR sequence located WO 01/21647 PCT/IB00/01470 77 towards the carboxyl terminal end was used as a control. Although the 170 antibody had an inhibitory effect on the oleate-induced 12SI-LDL binding in human PLC incubated without leptin, it did not prevent the leptin stimulatory effect on LSR activity (Fig. The stimulatory effect of leptin on LSR activity as lipoprotein receptor was seen not only in cells of human origin, but also in rodent hepatocytes. A brief, 30 min, preincubation of rat hepatocytes with 20 ng/mL mouse recombinant leptin at 37 °C increased oleate-induced 125- LDL binding to the cell surface in subsequent incubations at 4 OC (Fig. 6A), indicating that this stimulatory effect of leptin occurred rapidly. Northern blots showed that this leptin treatment did not increase mRNA levels significantly. Further, inhibition of cell protein synthesis (50 pM cycloheximide) did not suppress the stimulatory effect of leptin, while microfilament inhibitors pM cytochalasin B) reduced leptin stimulation by more than 80%. While not wishing to be limited by any particular theory, these results are consistent with the stimulatory effect of leptin on LSR activity resulting primarily from mobilization of a cryptic pool of receptors to the cell surface.

Figure 6B shows the additive stimulatory effect of leptin and oleate on the binding of chylomicrons to rat hepatocytes. This leptin and oleate-induced binding of chylomicrons to rat hepatocytes was suppressed by specific polyclonal anti-LSR antibodies (Fig. 6C). Thus, the stimulatory effect of leptin on LSR is not limited to LDL, but extends to TG-rich lipoproteins that are directly responsible for the transport of dietary lipid. The data show that physiological amounts of leptin acutely regulate the removal of dietary TG by the liver, and that in vitro, the same concentrations of leptin regulate LSR activity in hepatocytes while leaving that of other lipoprotein receptors unchanged.

The inhibition of the intestinal absorption of dietary lipids by leptin was also investigated. Overnight-fasted ob/ob mice were gavage-fed a high fat test meal. Immediately after the test meal (time 0 the mice were injected intravenously with 200 pL saline containing either no supplement, 0.5 pg recombinant mouse leptin, 2.5 mg lactoferrin, or a mixture of 0.5 pg leptin and 2.5 mg lactoferrin. Blood samples were taken at 2 and 3 h after the test meal, and plasma TG concentrations were measured (see Table, below). Values for these 2 time points were pooled and are presented as means SD of quadruplicate determinations obtained in 2 different animals for each condition p 0.02 (saline versus leptin; I p 0.01 saline versus lactoferrin; 0 NS (lactoferrin versus leptin lactoferrin)).

Table Effect of lactoferrin and/or leptin on the plasma lipid response of ob/ob mice Plasma TG 2-3 hours after test meal (mg/mL) Saline 1.04 0.08 WO 01/21647 PCT/IB00/01470 78 Leptin 0.79 0.1 Lactoferrin 2.02 0.26 Leptin Lactoferrin 1.96 0.42 1 The amplitude of postprandial lipemia is determined by both the rate of intestinal lipid absorption and the rate of lipid clearance. To distinguish between these two possible sites of leptin regulation, we used lactoferrin, a milk protein that inhibits the removal of dietary lipid by the liver (Huettinger Retzek, Eder, M. and Goldenberg, H. (1988). Clin. Biochem.

21,87-92). As shown in the Table, injection of lactoferrin in ob/ob mice caused a doubling of plasma TG measured during the postprandial stage. Further, leptin caused a decrease in postprandial plasma TG when injected without lactoferrin, but was unable to achieve a significant effect in mice simultaneously treated with lactoferrin. Although not wishing to be bound by a particular theory, this suggested that most of leptin's regulatory effect was due to stimulation of dietary lipid removal by the liver. Lactoferin has been shown previously to be an inhibitor of LSR at the concentration used (Yen, Mann, Guermani, Hannouche, Hubert, Hornick, Bordeau, Agnani, and Bihain, B.E. (1994) Biochemistry 33, 1172-1180; Mann, C. Khallou, Chevreuil, Troussard, A.A., Guermani, Launay Delplanque, Yen, and Bihain, B.E. (1995) Biochemistry 34,10421-10431).

The effect of leptin injection on the activity of lipolytic enzymes that are involved in the hydrolysis of plasma TG was also examined. Injections of leptin (50 pg/animal) did not significantly modify lipase activity released in serum ofdb''/db'" after heparin injections (Fig.

12). If anything, leptin decreased, although not significantly, the lipase activity when compared to the effect of administering the test meal alone. These data ruled out the possibility that leptin regulates postprandial lipemia by directly controlling the activity of lipolytic enzymes.

EXAMPLE 6: Comparison of the Effect of Human and Mouse Leptin To establish a link between leptin control of postprandial lipemia in mice and its stimulation of LSR in cultured cells, the species specificity in the ability of mouse and human leptin to activate LSR in cultured cells was utilized. Mouse leptin was more efficient than human leptin in stimulating LSR-mediated LDL degradation in primary cultures of rat hepatocytes (Fig. 7A); binding and uptake of 1sI-LDL followed a pattern superimposable to that of 2 I-LDL degradation. Conversely, human leptin was more efficient in stimulating LSR activity in human PLC cells than mouse leptin (Fig. 8B).

The effect of human (1 pg/animal) and mouse (0.25 tg/animal) leptin on plasma TG response of dbP"'/dbh"' mice was also compared. The data showed that human leptin slightly WO 01/21647 PCT/IB00/01470 79 reduced the postprandial plasma TG response (Fig. 7B, closed bar), but the effect did not reach statistical significance. This is consistent with the relative inability of human leptin to stimulate rodent LSR activity in cultured cells (Fig. 7A, closed bar). Mouse leptin injected at a 4-fold lower dose had a pronounced effect on postprandial plasma TG (Fig. 7B, hatched bar), consistent with its profound stimulatory effect on LSR in cultured cells (Fig. 7A, hatched bar). Thus, the effects of human and mouse leptin on postprandial TG response in obese mice paralleled their ability to stimulate LSR activity as lipoprotein receptor in cultured cells. Such species specificity has not been shown for the Ob-R.

EXAMPLE 7 Differential Effect of Mouse and Human Leptin and Leptin Peptide in Cells Species specificity has been observed with respect to leptin's ability to increase LSR activity in rodent or human liver cells (Fig. 8A and 8B). Mouse leptin increases LSR activity more in rat hepatocytes, and human leptin increases LSR activity more in human cells. In human cells the mouse leptin is inactive and almost approaches an inhibitory effect.

An internal seqment of the leptin polypeptide that is near the carboxy terminus was found to differ significantly in different species (See shaded area in Fig. 13). The mouse and human sequence of this segment was synthesized as a 22-mer peptide and tested for activity in cells (Fig. 9 10). The human peptide was agonistic for LSR activity in human cells, while the mouse peptide was antagonist for LSR activity in human cells. Thus, the human leptin peptide has a complete signalling capacity in human cells (Fig. In primary cultures of rat hepatocytes, both peptides increased oleate-induced LDL binding, though not to the same exten (at concentrations 50 glg/mL)t. However, there was an inhibitory effect on oleate-induced LDL degradation, indicating that these peptides do not completely mimic the activity of leptin in the rat system (Fig. EXAMPLE 8 Effect of Mouse Leptin or Leptin Peptide on the Post-prandial Response The apparent Kd of LSR for leptin is in the same range as that of the Ob-receptor, suggesting that the regulation of LSR activity by leptin could represent a physiologically relevant process. To address this issue, the variation in plasma leptin concentration that occurs after administration of a test meal to normal mice was measured. Leptin concentrations of 1.9 0.7 and 4.5 0.2 ng/mL (p<0.007, n=4) were measured before and 2 h after the meal. However, in normal mice, the postprandial increase in plasma TG remained small and transient, even when massive amounts of dietary lipid were provided by intragastric cannulation. This reflects the fact that in normal mice, the rate of lipid clearance is adapted to that of intestinal absorption.

Imbalance of this system appears to occur only in obese mice. However, dbp"/db" mice are not a satisfactory model to test the physiological effect ofleptin. The plasma leptin levels of WO 01/21647 PCT/IB00/01470 these animals are extremely high (86.7 12.2 ng/mL) and furthermore, do not detectably vary after administration of a test meal. Two hours after the test meal, leptin concentrations were measured as 86.6 18.9 ng/mL (NS, Therefore, ob/ob mice that lack leptin were used to test whether administration of a physiological dose of leptin modulates postprandial lipemia.

As seen in Fig. 11A, a single subcutaneous injection of 50 ng of leptin in ob/ob mice decreases the postprandial lipemic response. This injection caused a transient increase in plasma leptin concentrations up to 3.25 0.03 ng/mL at 2 h; baseline values were recorded 4 h after injection. The dose of leptin that is needed to control postprandial lipemia in ob/ob mice is 5-10 fold lower than those used in leptin-resistant db/db mice. In ob/ob mice, the signaling effect of leptin could result either from interaction with the Ob-receptor or the LSR.

A synthetic peptide with a sequence identical to that of mouse leptin between residues 117-138 was obtained and found to stimulate the oleate-induced binding of '"I-LDL in primary cultures of rat hepatocytes (Fig. 11 B, insert). Daily subcutaneous injections of 25 pg of this synthetic leptin peptide to ob/ob mice had no effect on the food intake over a 12 day period (7.6 0.4 g/day in ob/ob receiving saline and 6.7 0.3 g/day in ob/ob receiving peptide; n 4, NS).

Daily injections of 25 pg of mouse leptin caused a reduction of food intake to 4.7 0.5 g/day (n 3; p 0.003 versus controls). Thus, the synthetic peptide that activates LSR in vitro does not influence food intake by activating the Ob-receptor. Injection of 50 ng of this synthetic peptide reduced the postprandial lipemic response in ob/ob mice (Fig. 11B).

EXAMPLE 9: Relevance to Disease States The instant invention has shown that leptin regulates cellular functions in the absence of functional Ob-R. A myriad of peripheral regulatory effects of leptin have been identified and attributed to leptin signaling through the Ob-R, even when the targeted tissues lack the long isoform of the Ob-R, the sole isoform with a clearly established signaling capacity (Friedman, and Halaas, J.L. (1998). Nature 395, 763-770). The characterization of a leptin receptor distinct from the Ob-R and controlling the entry of exogenous TG into the liver opens the possibility that leptin controls other aspects of cell metabolism independently of the Ob-R. Although not wishing to be limited to a particular theory, one hypothesis is that leptin resistance is due to desensitization of the signaling pathway through which leptin binding to LSR leads to mobilization of the receptor to the cell surface.

Leptin regulation of the exogenous lipoprotein pathway opens new perspectives towards the understanding of the relationship between obesity, hypertriglyceridemia and cardiovascular disease. Indeed, accumulation in plasma of the residues of chylomicrons has been shown to increase the risk of cardiovascular disease due to the formation of atherosclerotic plaque (Karpe et al, 1998 Atherosclerosis 141, 307-314). Hypertriglyceridemia is also considered an WO 01/21647 PCT/IB00/01470 81 independent predictor of cardiovascular disease in obese subjects with Type II diabetes (Feeman, 1998 Ann. Intern. Med. 128, 73-74).

By increasing the contribution of the liver to the removal of plasma TG, leptin prevents deposition of dietary lipid in adipose tissue in excess of their FFA-releasing capacity. Thus the liver plays a critical but underestimated role in the pathogeny of obesity.

EXAMPLE 10: Molecular Modeling of an Active Leptin Fragment of the Invention The amino acid sequence for the human leptin fragment with activity is: NH 2 CHLPWASGLETLDSLGGVLEAS-COOH (SEQ ID NO:57; residues 117-138). The amino acid sequence of the mouse leptin fragment with inhibitory activity in the human system is: NH 2 CSLPQTSGLQKPESLDGVLEAS-COOH (SEQ ID NO:67).

A molecular dymamic assay (MD) was performed on both the human and the mouse 22aa peptides. MDs were performed under AMBER force field, in vacuo, with a dielectric constant proportional to 4r, a switched cutoff with inner radius of 10A and outer radius of 14A, a heating phase of 20ps from 0 to 300K by steps of 50K, and a production phase of 120ps at 300K.

At the end of the 120ps MDs, both peptides have lost their short helical part, and have shrunk to a more compact conformation.

The main difference between the human and mouse 22aa peptides in the packed conformations is the presence of a residue with higher accessibility (namely L133, before the 2 Glycines of the end sequence LGGVLEAS) in the human 22aa peptide.

In order to decipher which amino acid is important amoung the 126-129 amino acid residues, which differ significantly between human and mouse, the following in-silico combinatorial mutational assay was performed.

Each residue in positions 126-129 of the 22aa human peptide (conformation extracted from the human leptin) was mutated, resulting in 16 mutated peptide models. Each model was minimized until reaching an rms gradient of 0.1 Kcal/mol (within the AMBER force field).

Then, each minimized model was used as the starting conformation of ultra-short molecular dynamics (MD) assay (heating phase from OK to 300K of 20ps, and production phase at 300K of in vacuo, under the same conditions as described above). The final MD snapshots were reminimized, and the corresponding energies are given in the following HTML table, as well as the sequence of the spontaneously formed alpha helices.

Energies of 16 Mutated Human 22aa Leptin Peptides Central Sequence LD LE PE PD ET -87.4 -79.3 -83.9 -69.3 LDSLGG TPDSL WO 01/21647 (SEQ ID NO:42) -66.0

GLQTLDSIG

(SEQ ID NO:47) -82.5

SLGGVLEAS

(SEQ ID NO:50) -83.3

LGGVLEA

(SEQ ID NO: 53) PCTIBOOIO147O (SEQ ID NO:46) -65.4

TPDSLG

(SEQ ID NO:49) -83.3

GGVLE

(SEQ ID NO:48) -93.1

PESLGG

(SEQID NO:5 1) -68.0 -92.2 -92.2

PDSLGG

(SEQ ID NO:52 -85.2 -90.2 -84.2 Left column: first 2 aa residues of the mutated ETLD (SEQ ID) NO:40) human motif. First line: last 2aa residues of the mutated ETLD (SEQ ID NO:40) human motif. Information available in each cell: energy of the minimized 20 ps snapshot (Kcal/mol), and alpha helix sequence if present in the 2Ops snapshot. Peptides containing ETLD (SEQ ID NO:40; human motif) and QKPE (SEQ ID NO:41; mouse motif) are in italic.

Under these conditions, the EKLE (SEQ ID NO:43), EKPE (SEQ ID NO:44) and EKPD (SEQ ID NO:45) containing peptides are the most favorable ones and have an alpha helix.

QKPE (SEQ ID NO:41; mouse motif) and ETLD (SEQ ID) NO:40; human motif) containing peptides are the next favorable conformations, with an alpha helix for ETLD (SEQ ID Since the residue composition of each peptide is different, both composition and conformation energies form part of the comparison, and not only conformation energies.

Other peptides of the invention that can be tested in the assays described herein or other comparable assays for LSR agonistic or antagonistic activity include the following: Table Human Leptin Peptide Fragments iloito Seqenc SQEICIDiNME 117-138 CHLPWASGLETLDSLGGVLEAS FSEQ IDNO:57 122-143 ASGLETDSLGUVLEASGYSTE SEQ ID 127-148 TLDSLGGVLEASGYSTEVVALS SEQ ID NO:62 132-153 CjGVLEASGYSTEVVALSRGQGS SEQ ID NO:63 112-133 ASKSCHLPWASGLETLDSLGG SEQ ID NO:56 107-128 LHfVLAFSKSCHLPWASGLETL SEQ ID 102-123 ENLRDLLHVLAFSKSCHLPWAS SEQ ID NO:54 119-136 PWASGLETLDSLGGVLE SEQ ID NO:58 WO 01/21647 PCT/IB00/01470 121-134 WASGLETLDSLGGV SEQ ID NO:59 123-132 SGLETLDSLG SEQ IDNO:61 Table Mouse Leptin Peptide Fragments 117-138 SLPQTSGLQKPESLDGVLEAS EQ ID NO:67 122-143 TSGLQKPESLDGVLEASLYSTE SEQ ID 127-148 PESLDGVLEASLYSTEVVALS SEQ ID NO:72 132-153 DGVLEASLYSTEWALSRLQGS SEQ ID NO:73 112-133 AFSKSCSLPQTSGLQKPESLDG SEQ ID NO:66 107-128 LLHLLAFSKSCSLPQTSGLQKP SEQ ID 102-123 ENLRDLLHLLAFSKSCSLPQTS SEQ ID NO:64 119-136 LPQTSGLQKPESLDGVLE SEQ ID NO:68 121-134 QTSGLQKPESLDGV SEQ ID NO:69 123-132 SGLQKPESLD EQ ID NO:71 EXAMPLE 11: Inhibition of the Expression of Endogenous LSR Using Chimeraplasty Chimeraplasty experiments to inhibit the expression of cellular LSR are designed based on publications by Cole-Strauss et al. (Science 273 :1386-1389 (1996)) and Alexeev and Yoon (Nature Biotech. 16 :1343-1346 (1998)). The following Example is exemplary only. Other sites in LSR can be targeted using the same approach to achieve either inhibition of expression, or to change base pairs to study the importance of various residues (both protein coding and within regulatory regions, intronic, or 5' or 3' to the coding region) for LSR functioning in vitro and in vivo. Similarly, chimeric oligonucleotides can be designed to modify LSR amino acids either in the coding or non-coding regions in experimental animals and for treatment of diseases in humans.

There are two ATG codons in human LSR. The second ATG corresponds to the ATGs present in mouse and rat LSR. The first ATG is used as the start site for at least some of the forms at least some of the time, since the N-terminal antibody 81B is specific for this region of the LSR protein (See other Examples). Therefore, chimeric oligonucleotides were designed for the region after the first ATG and before the second ATG, and the region after the second ATG.

The first step was to identify regions of LSR where changing a single base pair results in the creation of a stop codon. Although there are three stop codons, TAG (amber), TAA (ochre) and TGA (stop), TGA is preferred for giving a complete stop (complete inhibition of LSR WO 01/21647 PCT/IB00/01470 84 expression). Two regions were identified (one after the first ATG and one after the second ATG) where changing a single base pair would result in a TGA stop codon, and chimeric oligonucleotides were designed for the appropriate sequences (Fig. Chimeric oligonucleotides are designed such that they will basically form a double-stranded sequence with two sets of 4T's at the bends and a GC-clamp (typically 5 bases in length) at one end and the mutated sequence and its wild-type complement forming the main part of the double-stranded part (typically 25 bases in length). Flanking the mutated sequence (typically 5 DNA bases) is 2'o-methyl RNA sequence (typically 10 bases on either side).

Primers and probes were also designed for these regions for use in an allelic discrimination assay (PE Applied Biosystems, <<Allelic Discrimination Using 5' Nuclease Assays> www2.perkin-elmer.com/ab/apply/dr/dralb4.html). The use of flourogenic probes in a nuclease assay combines PCR amplification and allele detection into a single step.

Hybridization probes for the endogenous and mutant forms of the allele are included in the PCR amplification reaction. The hybridization probes are cleaved by the 5' nuclease activity of Taq DNA polymerase only if the probe's target sequence is being amplified. By using a flourogenic probe, cleavage of the probe can be detected without post-PCR processing. The flourogenic probe comprises an oligonucleotide labeled with both a flourescent reporter dye (typically and a quencher dye (typically In the intact probe, the proximity of the quencher reduces the flourescent signal from the reporter dye. Cleavage liberates the reporter dye allowing an increase in its flourescent activity. The essence of the technique is that it can detect single nucleotide mismatches since these interfere with the ability ofTaq DNA polymerase to cleave the probe.

Probe placement is dictated by the location of the polymorphism. Generally, the polymorphic site should be near the center of the probe, since mismatches at the ends are not typically as disruptive to hybridization. A separate probe is synthesized for each allele, and each is labeled differently (FAM and TET or JOE, for example). The main criterion for probe selection is that it be long enough to hybridize at the annealing/extension temperature used in the PCR amplification. Calculation of the annealing/extension temperature is routine for those of ordinary skill in the art. Typically a probe Tm (melting temperature) of 65-67 C works well at an annealing temperature of 60-62 C. Therefore, the length of each probe is typically adjusted so that both probes have an estimated Tm of 65-67 C. In addition, there can be no G at the end, since a G adjacent to the reporter dye quenches flourescence somewhat even after cleavage.

The probes can be for either strand; the strand with more C's than G's generally performs better in the 5'nuclease assay.

Primers are chosen based primarily of estimated Tms as well as small amplicon size.

Primers with Tms of 58-60 C (approximately 5 C below the probe Tm) generally work well at WO 01/21647 PCT/IB00/01470 annealing/extension temperatures of 60-62 C. Generally, primers that are unstable at their 3' ends are preferred, as this seems to reduce non-specific priming. Therefore, primers with only one to two Gs and Cs within the last 5 nucleotides of the 3' end are preferred. In addition, primers should be placed as close as possible to the probe location without overlapping the probes. This generally results in amplicons of less than 100 bp, which is advantageous for PCR amplification success.

First ATG: Chimeric oligonucleotides. DNA is in capital letters; 2'o-methyl RNA is in small letters; mutated base is underlined: ATGCAACAGGACGGACTTGGAGTAGTrTTcuacuccaagTCAGTcuguugcauGCGCGTT TCGCGC 3' (SEQ ID NO:74) Allelic Discrimination Assay: Forward Primer: TGTCCACGTCGTTTACGCTC (SEQ ID Reverse Primer: TCCCACTTCCGTCCTTGTC (SEQ ID NO:76) Probes (endogenous/mutant): 3' CCTACTCCAAGTC(C /A)GTCCTGTTGCATT- (SEQ ID NO:77) Second ATG: Chimeric oligonucleotides. DNA is in capital letters; 2' o-methyl RNA is in small letters; mutated base is underlined): GACCCTGCCCTGTACCTACCTACCAGATGTTITcaucugguagGTTCAgggcagggucGCG CGT1TT 3' (SEQ ID NO:78) Allelic Discrimination Assay: Forward Primer: GTGGTGATCCTCTTCCAGCCT (SEQ ID NO:79) Reverse Primer: CCAGATGACGATGGGTfGC (SEQ ID Probes (endogenous/mutant): 5' ACCCTGCCCTG(T/A)CCTACCAGATGAC 3' (SEQ ID NO:81) The chimeric oligonucleotides are also made flourescently labeled to allow tests for transfection efficiency.

Following synthesis of the chimeric oligonucleotides and the primers and probes for the allelic discrimination assay, the flourescein-labeled chimeric oligonucleotides are transfected into PLC cells using standard methodology (other Examples), and the transfection efficiency determined by flourescence. The proportion of cells that are flourescent (successful transfection) is compared with the total number of cells by techniques that are standard in the art.

WO 01/21647 PCT/IB00/01470 86 If the transfection efficiency is low, various parameters of the transfection methodology may be modified to increase the transfection efficiency. These parameters are well-known in the art.

Following a successful transfection of the flourescently-labeled chimeric oligonucleotides, the unlabeled chimeric oligonucleotides are transfected into PLC cells, and the cells are sorted using FACS (flourescent activated cell sorter) after labeling cells with a first anti- LSR antibody followed by a flourescently-labeled second antibody that binds the first antibody using methods standard in the art. The first antibody can be the N-terminal specific 81B antibody to sort cells for LSR expression following mutation of the site after the first ATG, but needs to be a more C-terminal specific antibody (such as the 170 antibody (to mouse carboxy terminus) or 93A (to same region of human carboxy terminus)) to sort cells for LSR expression tested for creation of the stop codon and expression of LSR expression following mutation of the site after the second ATG.

The cells in both groups with the lower LSR expression are collected to enrich for cells with the stop codon in at least one of the copies of LSR. The cells are then cultured and checked for the presence of the stop codon mutations using allelic discrimination. An exemplary reaction set-up and procedure is as follows REAGENT FINAL CONC. (IL) TaqMan Buffer A lX mM MgC12 5 mM dATP 200 M dCTP 200 aM dGTP 200 pM dUTP 400 pM AmpliTaq Gold (5 U/pL) 1U 0.2 AmpErase UNG (1 U/pL) 0.25 U 0.25 DEPC H 2 0 2.55 TOTAL VOLUME 12.5 pL The primer concentrations can vary from 100 nM to 300 nM. Probe concentrations can vary from 50 nM to 200 nM. Template concentrations can vary from 0.1-100 ng/reaction.

STEPS

1. 50 C for 2 min.

2. 95 C for 10 min.

3. 95 C for 15 sec.

4. 58 to 65 C for one min.

5. hold at 4 C Repeat steps 3 4 for 40 cycles.

WO 01/21647 PCT/IB00/01470 87 Following testing, the cells are retransfected with the chimeric oligonucleotides and again sorted for LSR expression using FACS. The cells that are expressing the lowest amounts of LSR (or none) are selected, cultured to form a homogeneous population, and rechecked using allclic discrimination to identify cell clones that no longer express LSR. These cells can then be used in assays to study the role of the various LSR subunits and the interaction of compounds with particular subunits, as well as for screening for modulators of specific LSR activities (modulated by the different subunits, for example). In addition, the above-described techniques can be used on other cells, (including those in the ATCC databank and in animals or humans) to create other kinds of cells lacking LSR activity. As well as the uses as a research and compound screening tool, the technique is also useful for treatment of diseases related to obesity in vivo.

Chimeric oligonucleotides were also designed to specifically inhibit either the a subunit of LSR, or both the a and the a' subunits of LSR, by targeting either Exon 4 or Exon specifically.

Exon 4 Chimeric oligonucleotides. DNA is in capital letters; 2' o-methyl RNA is in small letters; mutated base is underlined): -TGGCTGAGCTCTTACCTGGTTTCATTTTtgaaaaccagGTCAGagctcagccaGCGCG

I

T

TTCGCGC 3' (SEQ ID NO:82) Allelic Discrimination Assay: Forward Primer: GAGCTCATCGTCCTTGGGAG (SEQ ID NO:83) Reverse Primer: AGTCTTCTATGGGCCCCGC (SEQ ID NO:84) Probes (endogenous/mutant): 3' CACCGACTCGAGA(A/C)TGGACCAAAAGTC (SEQ ID Exon Chimeric oligonucleotides. DNA is in capital letters; 2' o-methyl RNA is in small letters; mutated base is underlined): GGTTGTGGTATGCCTGGCTGCCTTCTTTTgaaggcagccAGTCAtaccacaaccGCGCGT TTTCGCGC 3' (SEQ ID NO:86) Allelic Discrimination Assay: Forward Primer: ACGCAGAGCTCATCGTCCTT (SEQ ID NO:87) Reverse Primer: GATGCCCAGGAGGAGGAAGA (SEQ ID NO:88) Probes (endogenous/mutant): 3' CAACACCATAC(G/T)GACCGACGGAA (SEQ ID NO:89) For both, use FAM as the dye for the endogenous nucleotide (A and G, respectively), and JOE as the dye for the changed nucleotide (C and T, respectively).

WO 01/21647 PCT/IB00/01470 88 EXAMPLE 12: Use of Zinc Finger Polypeptides for LSR Modulation A method for specifically binding DNA of choice and repressing or initiating its transcription has been described recently in WO 98/54311. The repression or initiation can be constitutive in the presence of the vector carrying the zinc finger, or it can be placed under the control of a small molecule switch, for example the TET system, where the expression of the repressor/initiator-bound zinc finger can be regulated. This is especially important in systems where complete absence of a gene at certain developmental stages, for example, is lethal, or where it's overexpression is toxic (Massie B, Couture F, Lamoureux L, Mosser DD, Guilbault C, Jolicoeur P, Belanger F, Langelier Y Inducible overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette. J Virol 1998 Mar,72(3):2289-96).

Zinc finger polypeptides are designed to specifically bind to LSR genomic DNA, and then are linked with the KRAB repressor to inhibit LSR expression. Sequences identified for use in making the zinc finger polypeptides are 1936 to 1927 of SEQ ID NO:1 TAG GGG TGA GCG GCG GGG (SEQ ID NO:91) 1947 to 1936 of SEQ ID NO:1 1946 to 1936 of SEQ ID NO :1 1956 to 1947 of SEQ ID NO :1 1956 to 1946 of SEQ ID NO :1 2304 to 2295 of SEQ ID NO :1 1778 to 1787 of SEQ ID NO :1 1787 to 1798 of SEQ ID NO :1 1946 to 1934 of SEQ ID NO :1 1934 to 1922 of SEQ ID NO :1 1740 to 1749 of SEQ ID NO: 1 GAG GGC TGG NNN TAG GGG TGA (SEQ ID NO:92) AGG GCT GGG NN TAG GGG TGA (SEQ ID NO:93) GTG GGA GCC GAG GGC TGG (SEQ ID NO:94) GTG GGA GCC N AGG GCT GGG (SEQ ID GCG GCG GCC GGG TGG GAG (SEQ ID NO:96) TG GCC GGA GCA GAT GGG (SEQ ID NO:97) GCA GAT GGG NN CCG GAA GGG (SEQ ID N098): AGG GCT GGG NNN AGG GGT GAG (SEQ ID NO:99) AGG GGT GAG NNN CGG GGA GGG (SEQ ID NO:100) AAG TGG GTC TCG GTT GCA (SEQ ID NO:101) The sequences to be bound by zinc finger polypeptides are provided to Sangamo, where the actual zinc finger proteins are synthesized and are linked to the KRAB domain, a transcription repressor (Pengue G, Calabro V, Bartoli PC, Pagliuca A, Lania L Repression of transcriptional activity at a distance by the evolutionarily conserved KRAB domain present in a subfamily of zinc finger proteins. Nucleic Acids Res 1994 Aug 11;22(15):2908-14), are synthesized. The DNA binding domain can also be linked to transcription initiators (such as VP16 Proceedings of the National Academy of Sciences USA 94 :5525 (1997)) or a small molecule switch system, that is used to turn on or off the zinc finger protein linked to the repressor or initiator. Examples of small molecule switches that are effective in cells and in animals include, the Tet system, RU486, and ecdysone.

WO 01/21647 PCT/IB00/01470 89 The zinc finger proteins are delivered as plasmids suitable for transfection into cells using standard techniques (Fugene, is a method of choice). The cells used include, but are not limited to, the human cell lines HepG2, PLC, Hep3B, C3A, and 293 and the mouse cell lines taoBpRcl, BpRcl, and Hepal-6. All cells are available from ATCC. Following transient transfection, the cells are tested for LSR expression and activity using standard techniques described in this application, that may include FACS analysis to look for LSR expression on the cell surface, quantitative PCR to look at whether the message is being made, and various binding, uptake and degradation experiments to study LSR activity.

Following a determination of which zinc finger proteins are the most effective in inhibiting LSR expression, stably tranfected cell lines are created, using the techniques described in this application. These cell lines are used to then study the activity of the subunits of LSR separately and in combination by co-transfecting them into the cells either stably or transiently, or by turning on and off endogenous LSR genes. These cell lines are the basis of assays for agonists and antagonists of LSR generally and the subunits separately and in any combination.

The zinc finger proteins are also provided as part of a supernatant associated virus, or retroviral adenovirus (for example adeno-associated viral These are effective gene transfer vectors for use in cells or in animals, as well as humans. Upon receipt, the AAV supernatant is amplified using techniques well-known in the art and examples are described in Xiao ct al. J. Virology 72 :2224-2232 (1998)) and can include the use of helper plasmids as described in Collaco et al (Gene (1999) 238:397-405). Following amplification, the supernatant is used to infect cells or preferably mice using standard techniques in the art some examples of which are provided by Snyder et al. (Nature Medicine 5 :64-69 (1999) and Teramoto et al. J.

Virol. 72 :8904-8912 (1998).

Following infection, the cells are tested as described above the mice are tested for effects on fasting and post-prandial levels of triglycerides, free fatty acids, cholesterol, leptin, glucose, insulin, and adipoQ (Acrp30, Apml) as well as fragments thereof, for example, before and after feedings as described herein. Similarly to plasmids, contructs in AAV gene transfer vectors can be co-infected. Thus, mice or cells can be co-infected with constructs containing cDNA encoding the a, or P subunits either alone or in combination to study their role in vivo and to test the effects of agonists/antagonists on specific subunits, or subunit combinations, in animals or cells.

LSR Zinc Finger Proteins WO 01/21647 PCT/IB00/01470 Sangamo's Universal GeneTools technology platform enables the rational design and rapid generation of highly specific ZFP transcription factors that can selectively recognize and regulate/modulate transcription of any target gene or DNA sequence. Expression of the ZFP's as fusions to activation (herpes simplex virus VP16) or repression (Kruppel-associated box A domain KRAB-A) domains allows transcription to be specifically up or down modulated within cells. Figure 25 contains a table with a summary of the five sets of plasmids encoding ZFPs targeted to the LSR gene. Each set contains the ZFP target sequence fused to the VP16 domain (NVF), or the KRAB-A domain (NKF). The sequences for the NVF versions of these plasmids are listed in Figure 26. These engineered ZFP's are being used for the functional analysis of LSR in both cell-based assays and in animal models.

Cell Based Assays: To determine the effect of these engineered on LSR expression, mouse hepatocytes were transfected and assayed for LSR mRNA by Northern analysis. Hepal-6 cells transfected with ZFP-NVF constructs, were harvested 24 and 48 hours post transfection for total RNA isolation (Qiagen RNeasy mini kit). Standard protocols were followed for Northern gels and blotting.

Blots were probed with the full length mouse LSR alpha cDNA (EcoRI fragment from pTracer clone) and G3PDH DNA (Clontech). Probes were prepared using Prime-IT I random primer labeling kit (Stratagenc) and 3 2 p dCTP. Quantitation of the Northern bands was done using Gel- Pro software.

Figure 27 shows an analysis of all 5 candidate ZFPs linked to VP 16. Only 2 of these plasmids, 5185 and 5186, exhibited any increase in expression, 6% and 16%, respectively, at 48 hours post-transfection. Since this increase was not very large, a more detailed analysis of these 2 ZFPs by Northern and QPCR was used to confirm the up-regulation of LSR by 5185 and 5186.

Hepal-6 cells transfected with ZFP-NVF constructs in triplicate, were harvested 24 and 48 hours post transfection for total RNA isolation (Qiagen RNeasy mini kit). Standard protocols were followed for Northern gels and blotting. Blots were probed with the full length mouse LSR alpha cDNA (EcoRI fragment from pTracer clone) and G3PDH DNA (Clontech). Probes were prepared using Prime-IT II random primer labeling kit (Stratagene) and 32p dCTP. Quantitation of the Northern bands was done using Gel-Pro software. The results show an average of 28% mRNA increase with 5186 and a 24% increase with 5185 (Fig. 28). It should be noted that there was no significant increase in LSR mRNA on either Northern at the 24-hour time point.

Since the Northern analysis is not quite as sensitive as QPCR, the transcriptional increase was confirmed using QPCR. Cells were harvested 48 hours post transfection for Total RNA isolation (Ambion RNaqueous Kit). RNA was then reverse transcribed to generate cDNA for PCR analysis. Primer and Probe sets directed toward the mouse LSR and control GAPDH WO 01/21647 PCT/IB00/01470 91 sequences were used to quantitate levels of transcription in ZFP transfected cells. As shown in Figure 29, QPCR results indicate a 41% increase in LSR transcription when Hepa 1-6 cells are transfected with ZFP plasmid 5186-NVF and a 30% increase with ZFP plasmid 5185-NVF.

These results indicate that both 5185 and 5186 plasmids were functioning in cells.

Binding-Uptake-Degradation (BUD) studies were used to assay the ability of these plasmids to increase the cells ability to process '2SI-LDL. Cultures of Hepal-6 mouse hepatocytes were transfected with ZFP's plasmids 24 hrs after plating. Cells were transfected with ljg plasmid/well in a 6well plate, using Lipofectamine (Gibco BRL) according to manufacturer's instructions. Forty-eight hours post transfection, Oleate-induced 1 2 5

I-LDL

binding, uptake, and degradation was measured as described herein.

Results of the BUD studies indicate increased binding and uptake of labeled LDL when Hepal-6 cells are transfected with ZFP's 5186-NVF and 5185-NVF when compared to control transfected cells. The data in figure 30 have been corrected either for total protein (30A-30C) or for 3-gal (30D-30F), which is a crude measure of the transfection efficiency. BUD data supports a role for ZFP 5186-NVF and 5185-NVF in the transcriptional activation of LSR and confirms a corresponding increase in functional activity.

The increase in LDL binding and uptake suggests an increase in expression of LSR at the cell surface. To prove this, cells transfected with the ZFPs were analyzed by Flow cytometry (FACs) Analysis. FACs analysis (described above) allows for direct estimation of the proportion of positive cells in a population, as well as an indirect measure of the level of receptor on the cell surface (mean fluorescence intensity).

Hepal-6 cells were transfected with ZFP-NVF constructs 5186 and 5185, along with control plasmids. Forty-eight hours post transfection, cells were analyzed for cell surface expression of LSR in the presence/absence of Leptin (20 ng/mL). Staining of Hepal-6 cells involved incubation with primary antibodies, generated in rabbits against mouse LSR NH2 terminal sequence CPDRASAIQ, or mouse COOH terminal sequence EEGHYPPAPPYSET, followed by detection with a fluorescent-labeled secondary antibody against IgG rabbit (Sigma).

Results indicate that in the presence of Leptin, Hepa -6 transfected with plasmid 5185- NVF had a 50% increase in the level of LSR on the cell surface when compared to controls.

While cells transfected with 5186-NVF had a 35% increase in LSR at the cell surface. These findings support a functional role for ZFP 5185-NVF and 5186-NVF in the transcriptional upregulation of LSR and concomitant increase of LSR on the cell surface.

Analogous experiments are used to assess the efficacy of ZFP-NKFs for repressing LSR transcription.

EXAMPLE 13: Retroviral Library Screening by FACS WO 01/21647 PCT/IB00/01470 92 In order to identify more genes involved in the regulation of LSR and in ligand signaling through LSR (leptin, Clq, AdipoQ (Acrp30, Apml), triglyceride-rich lipoproteins, etc) a retroviral library screening assay has been designed. In its most basic form, cells expressing LSR (PLC or HepG2, for example) are transfected with a retroviral library. Following sorting for expression of a marker protein, the cells are treated with a LSR ligand (leptin, for example) and assayed for LSR expression by FACS following staining with an antibody to LSR. Cells of interest, are those that either express more LSR or less LSR than is expressed following leptin stimulation of the same cells without the retroviral library.

The assay takes advantage of a retroviral vector developed by Lodish at the Whitehead Institute for Biomedical Research that takes advantage of the spectrum of expression levels of cloned cDNAs while simultaneously maintaining the high efficiency of retroviral gene transfer.

The vectors employ an encephalomyocarditis virus IRES (Jang et allJ. Virol. 62 :2636-2643 (1988)), followed by a quatitative selection marker, such as green fluorescent protein (GFP) or a cell surface marker protein, that are detectable by intrinsic fluorescence or by staining live cells with a fluorescent antibody, respectively (Fig. 14). Because expression of the two reading frames is strongly correlated FACS sorting based on the GFP or cell surface marker protein can be used to sort the cells for those cells expressing the unknown protein at a desired level-high, low, or moderate. For the proposed assay, the cells would preferentially be sorted for moderate expression, to allow a detectable, but not overwhelming effect The individual members of the gene library are placed upstream of the IRES (Fig. 14).

Genes of interest for screening for their effect on LSR expression on the cell surface include cDNA libraries from liver or adipose cells. Cells expressing LSR (such as Hep3B, HepG2, PLC) would be transfected by the library using standard techniques so as to achieve approximately I clone (gene) per cell. The cells would then be screened, and those with moderate expression of GFP would be selected for. Cells where endogenous LSR expression has been knocked out either by traditional methods, or using the Sangamo (zinc finger proteins) or chimeraplasty techniques described herein could also be used by co-transfecting various subunits of LSR (from 1-3 and any combination thereof), or in cells stably expressing recombinant LSR subunits, or combinations.

In the GLUT 4 system, described by Lodish (Whitehead), the GLUT4 gene was linked to 7 c-myc epitope tags and then GFP fused in frame at the carboxy terminus. This allows the quantity of the gene to be studied in the cell compartment where it is sequestered by comparing overall fluorescence with the GFP to cell surface fluorescence with anti-myc antibodies. A similar assay is envisioned for LSR where LSR could be fused to GFP (in this case the library would have to be linked to CD2 or CD4). Alternatively, the amount of LSR sequestered in a WO 01/21647 PCT/IB00/01470 93 cellular compartment could be determined using the 81B antibody, for example, and the amount of LSR on the cell surface could be determined using the 93A antibody, for example.

Once infected cells expressing moderate amounts of GFP are obtained, the cells can be treated with leptin, for example, (or any other LSR ligand of interest) and the difference in LSR levels in the compartment versus the cell surface, or simply on the cell surface can be determined by FACS (after antibody staining). Populations that have decreased LSR or increased LSR levels could be selected for. Optionally, the cells could be re-selected and then the retroviral DNA from the cells PCR'd and sequenced. Samples that appeared to be interesting by homologies or locations, for example, could then be cloned and re-transfected for further study.

This would allow the other genes that interact with this system to be discovered. The genes are likely to encode proteins whose modulation could have a direct impact on the regulation of obesity.

EXAMPLE 14: Effect of the Leptin Peptide in Mice with Congenital Lipodystrophy Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by a paucity of adipose tissue which is evident at birth and is accompanied by a severe resistance to insulin, leading to hyperinsulinemia, hyperglycaemia, and enlarged fatty liver (Seip et al Acta Pediatr Supp. 413 :2-28 (1996)). Leptin has been shown to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al.Nature 401:73-76 (1999)). These mice have extremely low levels of leptin in plasma. However, the authors do not link the effect of leptin with LSR. The instant invention includes the use of the the leptin peptides of the invention for treatment of lipodystrophy and for use in this mouse model.

Leptin peptide will be provided to transgenic mice expressing SREBP-lc436 in adipose tissue under the control of the adipocyte-specific aP2 promoter/enhancer (Shimomura et al.

Genes Dev. 12 :3182-3194 (1998)). The levels used are similar to those described for the ob/ob mice herein, a range around 50 ng per mouse. Leptin is provided daily for 12 days, either by injection, or using micro-osmotic pumps. Plasma glucose will be measured using a glucose (Trinder)-100 kit, plasma insulin by an anti-rat insulin radioimmunoassay (linco), and plasma leptin and triglyceride by standard methods described previously. A similar experiment is performed where the food intake is restricted to a level that is consumed completely by all animals.

WO 01/21647 PCT/IB00/01470 94 Example 15 Effect of Truncated Human LSR on Binding. Uptake Degradation of LDL Truncated forms of the LSR receptor were made and tested for their ability to function as either dominant positive increase the activity of the receptor) or dominant negative proteins interfere with the activity of the receptor), when over-expressed in cultured cells.

Materials: -Human LSR cDNAs a, a' and P from constructs made in pTracer CMV2.

-pcDNA/HisMax vector from Invitrogen -Appropriate restriction enzymes, T4 DNA polymerase I and Klenow, and T4 DNA ligase.

-Standard cloning procedures from "Molecular Cloning" by Sambrook et al.

-Follow construct plan (Figure Method of Cloning Testing.

1. Digest Human LSR plasmids with enzymes of interest under appropriate conditions.

Separate the appropriate insert fragment from the vector using agarose gel electrophoresis and Qiaquick gel extraction columns. Note: For constructs 1, 2, 3, 4a, 5, and 6 pTracerCMV2 LSR a was used as the source for the insert. For construct 4b, pTracerCMV2 LSR a' was used as the source for the insert. For 4c, pTracerCMV2 LSR P was used as the source for the insert.

2. Digest the pcDNA/HisMax vector in the appropriate reading frame with the enzymes of interest. Purify using agarose gel electrophoresis and Qiaquick gel extraction columns.

3. If necessary, treat insert fragments with Klenow DNA polymerase or T4 DNA polymerase I to blunt 3' overhangs. Purify DNA from the reaction using Qiaquick PCR purification kit.

4. Ligate inserts into vector according to Sambrook et al. using a 3-5 M excess of insert to vector.

5. Transform plasmids into competent E. coli- XLlblue from Stratagene. Follow manufacturer's instructions.

6. Isolate colonies with correct plasmids by either PCR or Qiagen miniprep analysis.

7. Verify correct clones by having them sequenced to ensure that they are in the proper reading frame and that there are no amino acid changes.

8. Grow and harvest DNA from large-scale cultures using Qiagen endotoxin free maxi preps.

9. Analyze constructs by transfecting them into human cells and assaying LDL binding using the standard BUD protocol.

BUD Assay Materials: -DNA from LSR truncated constructs at approximately 1 mg/mL.

-Lipofectamine Plus transfection reagent Life Technologies Cat. No 10964-013 -PLC cells plated at 0.3 x 106 cells/well in a 6 well plate.

WO 01/21647 PCT/IB00/01470 '2I-LDL mM suramin (70 mL PBS per 1 g suramin) -100 mM oleate in isopropanol, freshly prepared from a 400 mM stock solution -DMEM (without CaCI,) containing 0.2% BSA, 5 mM Hepes, 2 mM CaCI 2 pH 7.5, and 3.7 g/L NaHCO 3 (this media should be prepared before the experiment, stored at 4 0 C, and used for up to 1 week) PBS, pH 7.4 PBS containing 0.2 BSA 0.1 N NaOH containing 0.24 mM EDTA BUD Assay Methods: 1. Cells (adherent) in 6-well plates seeded at 3 x 10 5 cells 3 days prior to the BUD.

Transfcct the cells using lipofectamine plus reagent, according to the manufacturer's instructions, the day after the cells are seeded. Confluence should be between 50-80% when transfected.

Let cells go about 48 hrs (2 days) after transfection before BUD analysis.

2. Wash cells once with PBS (room temperature), 2 mL/well 3. Add DMEM/0.2% BSA (950 pL) 4. Add oleate, (0 to 1 mM oleate, e.g. 0, 0.1 mM 0.2 mM, 0.5 mM, 0.8 mM and 1 mL, from 100 mM stock) never exceed 10 pL isopropanol per mL DMEM It is necessary to include wells with no oleate as a control for background. This control allows one to calculate the amount of oleate-induced 25 1-LDL metabolized.

Add appropriate concentration of 2 I-LDL to each well (50 pjL of each dilution).

6. Incubate cells for 90 min to 4 hours at 37 OC in a CO 2 incubator. In these experiments, 3 hrs was the incubation time.

7. Transfer media from wells into 5 mL polycarbonate tubes. Store at 4 °C overnight for degradation analysis (see below).

8. Wash cells at 4 OC (on ice): Wash 2 times consecutively with ice-cold PBS/0.2% BSA Wash once with ice-cold PBS/0.2% BSA Wash 2 times consecutively with ice-cold PBS 9. Add 1 mL/ well 10 mM suramin and incubate at 4 °C for 1 hour.

Remove suramin into gamma counter tubes, and count for radioactivity. This represents the amount of 2 I-LDL bound to the cell surface.

WO 01/21647 PCT/IB00/01470 96 11. Add 0.1 N NaOH/0.24 mM EDTA (ImL/well) and incubate at room temperature for a minimum of 30 min. to lyse the cells.

12. Recover the cell lysates into gamma counter tubes and count for radioactivity. This represents the amount of 2 'I-LDL internalized. Alternatively, the suramin step may be omitted (LSR as leptin receptor) and the cells lysed immediately after washing. This would represent the amount of cell-associated '1 5 I-LDL or '251- leptin.

13. After cell lysates have been counted, determine the protein concentration per mL so that data can be reported as ng '12I-LDL bound/mg of total protein. Protein is determined using the BCA assay from Pierce according to the manufacturer's instructions. Alternatively, data can be corrected for P-Gal units by transfecting extra wells and collecting them for the P-Gal assay at the time the BUD is done. For this protocol, see P-Gal protocol, below.

Degradation of '2I-LDL After leaving overnight at 4 OC, add 1 mL ice-cold 40% TCA to the pre-cooled media.

Do not Vortex.

Incubate I hour at 4 0

C.

Centrifuge at 3000 rpm (Beckman Allegra centrifuge), 30 min 4 oC. (If the precipitate is floating, it is necessary to break the air-water interface by gently shaking the tubes before pelleting.

Transfer I mL supernatant to 5 mL glass tubes.

Add 40 pL 30% H 2 0 2 and vortex briefly.

Add 1 mL chloroform and vortex briefly. Let tubes sit for 15 minutes to allow separation of the 2 phases.

Transfer 0.5 mL to gamma counter tubes, and count for radioactivity.

For the calculation of the amount degraded, the dilution factor is 4.16. Corresponding plates without cells serve as controls to define the level of the background.

B-gal Assay Transfect cells with test construct the amount of p-gal expressing plasmid.

Harvest cells in lysis buffer (250 uL/well of a 6 well plate). Pull through a syringe several times before transferring into an eppendorf tube.

Freeze cells at -80 OC until ready to perform the assay.

Thaw cells of interest and spin at 14K in a microfuge at 4 OC for 5 min.

Transfer 10 iL of each lysate to a clear PP 96 well plate: WO 01/21647 WO 0121647PCTIBOO/01470 Example I 4 5 6 7 89 110 1112 A Blank Blank empty Sample Sample Sample Etc piL j2_pL 2l10 .iL 2 10 gL 2 104L B Control Control empty Sample Sample Sample *100 i *l100 .LL 3 V1L 3IOL 3 10~ 1 _1, C Sample Sample Sample Sample Sample Sample 1_ 10OLiL 1 10 L 1 10 p .4 410gtL 14 10 L 4 10 1 iL Control =reference standard #Blank reaction buffer only Add Fluo-Reporter P-gal substrate (Molecular Probes cat #F-2905) to P3 -gal reaction buffer.

(275 il, CUG substrate [component A] to9.73 mL of reaction buffer) NOTE: need 10 mnL for a 96 well plate, but if you don't use it all it can be stored at -20 *C for at least 6 months.

Add 100 p1. of Reaction buffer with substrate to each well.

Incubate at room temp. for 30 min.

Add 50 iL of stop mix (0.2 M Na 2

CO

3 Read on Cytoflour plate reader with 1 excitation at 360 and emission at 460. Gain should be set around l3-gal reaction buffer [Final1] M NaPhosphate pH 7.3 40 mL 0.IM I M MgCI, 0.2 mL 1 mM 14.3 M P-mercaptoethanol 629 il, 45 mM ddH 2 O 159.171 mL 200 mL Lysis Buffer: [Final] Buffer II [Final] I M Tris-Ac pH 7.8 50 ML 100trm I MMgAc 5 mL 10 mM M EDTIA I ML 1mHM ddH 2 O 43 m 500 mL Results of BUD Assa WO 01/21647 PCT/IB00/01470 98 Addition of the C-terminal portion of LSR increased 2

I

5 -LDL binding uptake and degradation in PLC cells (Fig. 16). 2 SI-LDL degradation is increased almost 2 fold at mM oleate. Data in this experiment is corrected for protein only. The transfection efficiency was not monitored. All points were done in triplicate. In a separate experiment, addition of the C-terminal portion of LSR also increased '2SI-LDL binding uptake and degradation in PLC cells (Fig. 17). 2 5I-LDL degradation was increased 2-3 fold at 0.5mM oleate. Data in this experiment was corrected for transfection efficiency only. All points are in triplicate.

The C-terminal portion of LSR from AA353 to 650 (the last AA) as well as the Cterminal portion from AA 353 to 541 are able to increase the binding, uptake and degradation of '1I labelled LDL in vitro (Figures 16 17). The increase is on the order of 2-3 fold for all 3 measurements when corrected for transfection effeciency using the P-Gal reporter as a carrier in the test DNA. The increase in LDL metabolism is still on the order of 2 fold when data are corrected for total protein, depending on the oleate concentration. These constructs can be cloned into a vector to allow expression and testing in vivo for this dominant positive effect in animals using methods well known to those in the art.

Example 16 LSR Gene Expression in Liver and Brain of Lean and Obese Mice LSR gene expression was determined by quantitative PCR (QPCR) in liver and brain tissue of 7 different mouse models: normal and high fat diet-fed C57BU6J mice (C57), C57BL6/J ob/ob (ob/ob), C57BLK/S, C57BLK/S db/db (db/db), NZB and NZO mice. The normal diet was obtained from Harlan Teklad (Teklad Certified LM-485 mouse/rat 7011C), the high fat diet, also called cafeteria diet was from Research Diets (D12331, Rat Diet 58 kcal fat and sucrose). The cause of obesity in the different models is high fat diet in the obese C57 mice, leptin deficiency in ob/ob mice, deficiency in functional leptin receptor in db/db mice. The cause of obesity in the NZO mouse is currently unknown (Lit C57BLK/S and NZB mice are both lean and were used as controls since they represent the corresponding background strain of db/db and NZO mice, respectively.

The qPCR results for the different LSR levels in the livers of different mouse strains are supported by immunohistochemistry result using methods well-known to persons of ordinary skill in the art.

Reverse Transcriptase Polymerase Chain Reaction Liver and whole brain were isolated from mice following perfusion with ice-cold saline containing 10 mM EDTA. Tissues were stored in RNAlater (Ambion, Austin) at 4 OC for 1 day and then at -20 oC. Liver total RNA was isolated using RNAqueous (Ambion, Austin) following the manufacturer's protocol. The amount of RNA was determined by absorption at 260 nm. The WO 01/21647 PCT/IB00/01470 99 quality of the isolated RNA was verified by the ratio 260/280nm (between 1.9 and 2.1 is good) and by denaturing agarose gel electrophoresis.

RNA was reverse transcribed to cDNA using oligo dT plus an LSR specific primer and Superscript II (Gibco BRL) according to manufacturer's instructions. The LSR specific primer is in exon 6 of the LSR gene (5'ACGCATGGGAATCATGGC; SEQ ID NO:90). Plasmids containing mouse LSR-alac'/ sequence were obtained by cloning RT-PCR products produced from mouse liver total RNA into pGEM-T easy (Promega). The sequence of the plasmid was confirmed by cycle sequencing on a ABI Prism 377 DNA Sequencer.

Quantitative PCR was performed on a ABI Prism 7700 Sequence Detection System using TaqMan technology (PE Biosystems). TaqMan assay primers and probes were designed using Primer Express software (PE Biosystems) and were synthesized by Genset, La Jolla. Each probe was double labeled with the fluorescent reporter dye 6-carboxyfluorescein (FAM) covalently linked to the 5' end of the probe and the quencher dye 6-carboxytetramethylrhodamine (TAMRA) attached to the 3' end. Uracil-N-glycosylase technology (PE Biosystems) was used to prevent contamination with PCR product.

PCR was performed using the following reagent concentrations 25 mM MgC12, dNTPs at 200 pM, except for dUTP at 400 pM, I U of AmpliTaq Gold, 0.25 U AmpErase UNG.

Primers were added at 300 nM and probes at 200 nM concentration. The forward and reverse GAPDH and LSR primers used are shown in Table 1. PCR reaction conditions were 50 °C for 2 minutes, 95 °C for 10 minutes, followed by 40 cycles at 95 OC for 15 seconds and 1 minute at OC. PCR was performed in 96 well reaction plates with optical caps and fluorescence was continuously followed for each reaction. cDNA corresponding to 15 ng of total RNA were used per PCR reaction.

Quantification of LSR expression was obtained using a standard curve of the corresponding LSR plasmid covering a concentration range between 5x 10 6 and 5x10'° 0

M

(approximately 106 to 102 copies). A standard curve of mouse (C56BL/6J) total liver RNA between 200 and 0.1 ng RNA was used to determine relative levels of GAPDH expression.

Amplification plots were analyzed using SDS software (PE Biosystems).

Table 1 PCR primers and probes used to determine the expression level of mouse GAPDH and mouse LSR isoforms.

Target Forward Primer Reverse Primer Probe Gene GAPDH AACGACOOCTTCATIGA CTITCCATCiTOGGCCIT ACTCACGGCAAATTCA/

I

~CGGC

CCrC G ACAG WO 01/21647 PCT/IB00/01470 LSR GGCAGGAGAATCACCAT complete CACA LSR alpha GCCCfTGGAAGATIGGC TCr LSR alpha' ACCAGGGCAGGAGAAT

CACC

LSR beta TTIUICITGTITATGCTG

CTGG

GATCiTTGGCLGAGACC

ACG

ATGCT3GCACACCMA

GGT

GGAGGAAGAAGAGGAG

GCTTG

CAGGAGAGAGGIGGT

ATAGATGG

I

IUUI UULGAGCOU TC GAGCA

GAG

CCAGTGCIUr=CCCACACCTGC

T

AGCTCATrIU=CrATD=GCT CTnTG AGCAGOXACCrCAGGTGGCC

AA

I

Quantification by TaqMan technology is based on determining the threshold cycle of amplification, which was determined for each unknown sample and for the standard dilutions using 0.1 fluorescence units as a threshold (maximum fluorescence The amount of unknown cDNA was calculated using the corresponding standard curve. LSR expression was given as absolute copy numbers and also normalized for GAPDH expression (by dividing the determined absolute copy number by the relative level of GAPDII for each individual animal).

Each determination was done in triplicate and was repeated at least once; very similar results were obtained.

All data were confirmed by standard Northern analysis. 16pg total RNA was pooled from 4 mice per group and tissue and analyzed by Northern. Although this type of analysis is semi-quantitative at best and LSR isoforms can not be differentiated, relative levels of gene expression show the same trends as measured by QPCR.

Results LSR Expression in Liver Table 1 LSR gene expression in liver of lean and obese mice (copy numbers in 15ng total liver RNA) LSR-alpha LSR-alpha' LSR-beta LSR GAPDH LSR total (sum of isoforms) C57 normal ave 93966 110334 18454 222754 2.8 281654 SEM 21760 16682 2790 39779 0.4 83220 ave 42.2% 49.5% 8.3% SEM 2.5% 2.4% 0.3% C57 obese ave 82814 44084 17280 144177 6.0 161206 SEM 12274 8073 2344 22521 1.7 21161 ave 57.4% 30.6% 12.0% SEM 1.2% 1.3% 0.4% C57 ob/ob ave 49898 51056 21126 122079 9.1 120026 SEM 5928 10469 1758 15113 1.0 32474 ave 40.9% 41.8% 17.3% SEM 0.7% 4.2% 3.9% C57BLK/S ave 49029 68379 41340 158749 3.9 163060 WO 01/21647 WO 0121647PCT/EBOO/01470

SEM

ave 3862 30.9% 1.3% 3721 43.1% 1.6% 2043 26.0% 1.8% 5903 0.4 94537 C57BLK/S ave 30625 48504 18683 97811 9.2 79745 db/db SEM 1953 12021 3123 10819 1.0 26413 ave 31.3% 49.6% 19.1% SEM 1.7% 7.0% 5.4% NZB normal ave 98455 387287 54079 539822 3.1 588656 SEM 4.446 13253 6740 21241 0.7 27993 ave 18.2% 71.7% 10.0% SEM 0.6% 0.8% 0.9% NZO obese ave 57497 225574 23377 306448 1.8 333271 SEM 4595 11767 1091 15948 0.3 11416 ave 18.8% 73.6% 7.6% SEM 0.9% 1.1% 0.2% LSR Expression in Brain of Lean and Obese mice Table 2 LSR gene expression in brain of lean and obese mice (copy numbers in O ng total liver RNA) LSR-alpha LSR-alpha' LSR-beta LSR GAPDH LSR total (sum of isoforms) C57 normal ave 1192 6443 7731 15365 36.2 10653 SEM 155 1512 443 1717 3.0 1933 ave 7.8% 41.9% 50.3% SEM 0.5% 6.0% 6.3% C57 obese ave 1496 10472 7418 19387 20.8 14118 SEM 155 1295 716 1998 5.7 805 ave 7.7% 54.0% 38.3% SEM 0.5% 1.9% 2.2% C57 ob/ob ave 1293 6502 6158 13954 34.2 14034 SEM 190 797 475 863 5.2 1939 ave 9.3% 46.6% 44.1% SEM 1.0% 3.4% 4.4% C57BLK/S ave 1918 5585 6456 13958 26.7 10458 SEM 206 354 1024 1087 5.3 980 ave 13.7% 40.0% 46.3% SEM 1.7% 2.8% 4.2% C57BLK/S ave 1834 5195 8189 15217 35.0 10912 db/db SEM 199 297 789 1117 4.5 670 ave 12.0% 34.1% 53.8% SEM 0.7% 2.0% 1.4% NZB nornmal ave 654 1019 5463 7135 17.0 4430 SEM 159 321 929 1051 4.7 926 ave 9.2% 14.3% 76.6% SEM 1.7% 5.1% 6.8% NZO obese ave 168 320 2715 3202 13.4 1446 SEM 112 52 37 1638 4.5 1008 ave 5.2% 10.0% 84.8% WO 01/21647 PCT/IB00/01470 102 SEM 12.9% 5.6% 16.8% C57BL6/J. C57BLK/S, db/db, ob/ob Mice LSR expression in the liver of obese animals is significantly lower than in lean control animals (Fig. 18). In general, the expression of LSR in brain tissue is much lower than in liver.

However, unlike in liver, obesity does not cause further downregulation (Fig. 21).

No significant differences in isotype patterns were found in liver samples from the different mouse models. LSR alpha and alpha' contribute equally and account for almost all of the total LSR expression. LSR beta contributes only a small percentage (Fig. 19 and Fig. In contrast, LSR alpha' and beta are the major contributors to overall LSR expression in brain, accounting in equal proportions for about 90% of total LSR message. No significant levels of LSR alpha were seen in any of the studied models (Fig. 22 and Fig. 23).

The downregulation of LSR seems to be strongly associated with obesity independent of the cause of obesity (dietary as well as different genetic defects are the causes in the used models). One might expect that upregulation of liver LSR expression in obese individuals would be beneficial.

NZB and NZO Mice LSR expression in liver tissue of NZB mice is 2-fold higher than in normal C57 mice.

Obesity (in the NZO) again leads to strong downregulation, however, this level is still significantly higher than in other obese mice (Fig. 18). In contrast, LSR expression in the brain of NZB and even more so in brain tissue of NZO, is significantly lower than in the other models (Fig. 21).

Distribution of LSR isotypes in NZB and NZO mice was very different from the previously described 5 models. The dramatic increase in liver LSR expression seen in NZB (and in NZO) mice was found to be mainly LSR alpha'. This form accounted for 80% of total LSR (Fig. 19 and Fig. 20). The complete opposite was seen in brain tissue. NZB mice have very low expression of LSR alpha and alpha' with LSR beta being the dominant isoform. This picture is even more pronounced in NZO mice. Brain LSR in these animals is almost exclusively LSR beta and some animals had virtually no alpha or alpha' expression (Fig. 22 and Fig. 23).

The fact that NZO mice respond to intracerebroventricular injection of leptin but not to peripheral injection (Halaas JL, et al., Proc.Natl.Acad.Sci. USA, 94, 8878-8883, 1997) suggests a transport defect. Since LSR alpha' has been shown to bind leptin, and since LSR alpha' levels are reduced in NZO mice, the implication is that the genetic defect in NZO mice causing obesity might be deficiency in brain LSR alpha' expression resulting in non-functioning leptin transport across the blood brain barrier. This conclusion is further supported by the discovery that some WO 01/21647 PCT/IB00/01470 103 NZO mice that do not become obese have LSR alpha' expressed at significant levels in brain.

Example 17 Effect of a Ser 4 Asn substitution on LSR activity in human hepatocytes Previously, we described a frequent (allele frequency 12%) G A mutation of cDNA base pair 1088 (LSR exon which results in a Ser "Asn mutation at amino acid position 363, presumably in the extra-cellular domain of the receptor.

In a group of 34 obese adolescent girls, this coding mutation significantly increased fasting and postprandial plasma triglyceride response to a high fat test meal. In a larger population of 154 obese adolescent girls, the same coding mutation significantly and selectively influenced fasting plasma triglyceride levels and increased 3.5 fold the risk of hypertriglyceridemia. This data suggested that LSR plays a significant role in the clearance of triglyceride-rich lipoprotcins. Interestingly, even individuals heterozygous at this locus showed the effect.

An in vitro model was obtained after sequence analysis of LSR in 2 cell lines, PLC and HepG2, revealed that PLC cells are homozygous for the G allele, while HepG2 cells are heterozygous, having both the G and A.allele.

Methods: The oleate-induced 2 I-LDL binding, uptake and degradation was measured in HepG2 and PLC according to the method described previously (Bihain, and Yen, F.T.

(1992). Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia.

Biochemistry 31, 4628-4636.). Briefly, confluent monolayers of cells were washed once in phosphate buffered saline (PBS), and then incubated 3 h at 37 OC with increasing concentrations of oleate (as indicated) and 20 pg/mL 12SI-LDL. At the end of the incubation, cells were placed on ice and washed twice with PBS containing 0.2% BSA, once with the same buffer, and then twice with PBS alone. The amounts of 2 I-LDL bound, internalized and degraded were then measured according to the method of Bihain, and Yen, F.T. (1992). Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 31, 4628-4636.

Results: The PLC cell line displayed a much greater capacity to bind, internalize and degrade 125- LDL in the presence of increasing concentrations of oleate, as compared to the HepG2 cell line (Fig. 24). This is most marked in the degradation. The decrease in degradation observed with mM oleate concentrations is thought to be due to the accumulation of oleate as triglycerides WO 01/21647 PCT/IB00/01470 104 in the cells. This increase in lipid in the cells decreases proteolytic degradation at the lysosomal level.

Quantitative PCR and facs data indicates that LSR expression is almost 50% higher in HepG2 cells than in PLC cells. This would be consistent with the notion of compensation for the lower activity of the receptor in the cells.

These in vitro data suggest that a person with a G/G genotype (hence Ser) would display a greater ability to clear triglycerides during the postprandial stage as compared to one with a G/A genotype. Since we have postulated a rate-limiting role of LSR in the removal of dietary lipid, these data could explain the significant association found between low postprandial triglyceride levels and G/G genotype. In contrast to G/G homozygotes, G/A heterozygotes with lower LSR activity would have a lesser capacity of removing dietary lipid, thus increasing their time in the circulation. This would in turn cause a change in the partitioning of lipid between the liver and the adipose tissue, leading to a greater deposition of fat in the adipose tissue.

This example indicates the potential use of this polymorphism, as a marker to detect people with a propensity towards obesity. It also supports the hypothesis that LSR is a potential pharmaceutical target for the development of compounds aimed at targeting lipids away from the adipose tissue and towards the liver.

Example 18: Leptin Transport through the Blood Brain Barrier Human leptin transport through the blood-brain barrier (BBB) is studied using an in vitro model (Dehouck, et al J Neurochem 54:1798-801, 1990). This model closely mimics the in vivo situation with regard to the selective passage of nutrients and drugs through the cerebro-vascular endothelium. The presence of tight junctions that prevent non-specific diffusion, the expression of specific receptors such as LDL receptor and transferrin receptor, and the expression of Pglycoprotein in brain capillary endothelial cells in vitro demonstrates that this model is a useful system to study the selective transport through the BBB. Briefly, this model consists of a coculture of bovine brain capillary endothelial cells (ECs) and rat astrocytes (Figure 31). The astrocytes are seeded on the plastic of a six-well dish and grown for 3 weeks. A collagen-coated filter is then set in each dish and bovine ECs are plated on the upper-side of the filter. ECs form a confluent monolayer in 5 days and they are used for experiments after 16 days ofcoculture with astrocytes.

Methods Leptin transcytosis: Experiments were performed on brain capillary endothelial cells in coculture with astrocytes for 16 days. On the day of the experiment, ECs were transferred to a clean 6-well plate containing 2 mL of Ringer-Hepes buffer (see, Fig. 32). At time 0, 1 mL WO 01/21647 PCT/IB00/01470 105 Ringer Hepes containing '"I-leptin was placed in the upper compartment. After 30, 60, 120, or 180 min incubation at 37 oC on a rocking platform, the insert was transferred into another well of a six-well plate to minimize the possible passage of substances from the lower to the upper compartment. At the end of the experiment, the amount of radioactivity of each well was counted. The transcytosis was performed over 3 h with 1) 10 ng/mL 12 5 I-leptin (10,000 dpm/ng), 2) O1ng/mL 12 5 1-leptin 1 pg/mL of cold leptin, 3) 10 ng/mL 25 1-leptin 50 ug/mL peptides or 4) 10 ng/mL 25 I-leptin 2 mg/mL lactoferrin. The synthetic peptides studied include the human (HP) and mouse (MP) leptin peptide fragments CHLPWASGLETLDSLGGVLEAS (SEQ ID NO:57) and CSLPQTSGLQKPESLDGVLEAS (SEQ ID NO:67), respectively.

Sucrose and inulin permeability studies: The 1 4 C]-sucrose (342Da) and ['H]-inulin (57000 Da) are hydrosoluble molecules which pass through the BBB through non-receptor mediated processes. The transport is nonspecific and primarily through tight junctions. These serve as markers for the integrity of the BBB and hence toxicity of the added compounds on the cerebral endothelium.

After 16 days ofcoculture, permeability studies were performed as described in Figure 32. On the day of the experiment, ECs were transferred to a new 6-well plate containing 2 mL of Ringer-Hepes. At time 0, 1 mL Ringer Hepes containing ['4C]-sucrose, ['H]-inulin and cold leptin were placed in the upper compartment. Sucrose and inulin permeability studies were performed in the presence of 10 ng/mL leptin, 5 ug/mL leptin, 10 pg/mL leptin or without leptin as a control. The effect of peptides was also tested by the addition of 10 ng/mL leptin+50 pg/mL mouse peptide 10 ng/mL leptin+50 pg/mL human peptide or 10 ng/mL leptin+2 mg/mL lactoferrin (lacto). At the end of the experiment, an aliquot from each well was placed in a scintillation vial, and radioactivity was determined.

The transport of molecules through the endothelial monolayer was determined for each time point as passage: passage of radiolabelled molecule through the endothelium: dpm found in the lower compartment at a time point divided by the initial dpm found in the upper compartment: transport at 30min (lower dpm t30 upper dpm)* 100 Results Figure 33 shows an increased transport ofradiolabelled leptin over time through the endothelium monolayer after 16 days of coculture. The addition of unlabelled leptin reduced the amount of leptin by approximately 30%, indicating that there is a specific component involved in the transport of leptin across the EC monolayer. A higher concentration of unlabelled leptin is needed to decrease the effect of nonspecific processes. The specific component involved in leptin transport is associated with the complete differentiation and formation of the BBB.

WO 01/21647 PCT/IB00/01470 106 Lactoferrin, an inhibitor of LSR, significantly inhibited the amount of leptin transported.

The mouse leptin peptide fragment had no significant effect on leptin transport. However, the addition of human leptin peptide fragment caused a significant increase in the amount of leptin transcytosis. This same peptide fragment increases LSR activity in human hepatocytes.

The integrity of the BBB was tested using sucrose and inulin (Figure 34A and 34B). It is clear that the integrity of the BBB was not significantly compromised by the addition of leptin, the peptides, or lactoferrin. Hence, we can conclude that the trancytosis measured in Figure 33 represents active processes, and is not due to disintegration of the EC monolayer.

Thus the invention is drawn to inhibitors and activators of LSR as a means for controlling the transport of leptin across the blood brain brain. Agents directed towards activation or inhibition of brain LSR regulate leptin transport into the CNS where it acts as satiety factor.

While preferred embodiments of the invention has been illustrated and described, it will be appreciated that various changes can be made by one skilled in the art without departing from the spirit and scope of the invention.

Example 19: Effect of Longterm Exposure to High Levels of Leptin on LSR Activity Human liver cells preincubated with 200 ng/mL human recombinant leptin for 24 h had a markedly reduced LSR activity (Fig. 35A, as compared to those not preincubated with leptin (Fig. 35A, Lcptin retained its ability to acutely increase oleate-induced '2SI-LDL binding to LSR in a subsequent short incubation (Fig. 35A, However, the maximal stimulatory effect was reduced by about 50%, and was achieved only with higher leptin concentrations (100 ng/mL). In hepatocytes preincubated for 24 h with high doses of leptin (200-400 ng/mL), a decrease of hepatocyte LSR mRNA relative to GAPDH was observed, as compared to control cells (Fig. Although not wishing to be limited by any particular theory, these data suggest that the consistently elevated leptin levels in db/db mice cause a decrease in LSR expression, as well as cause a reduction in leptin's ability to acutely stimulate the receptor. This, and the fact that plasma leptin did not increase in dbP"/db p after the test meal could explain the massivelyelevated postprandial lipemic response observed in this strain. However, because leptin signaling to LSR proceeds independent of the Ob-R, acute increase in plasma leptin concentrations obtained with injection of 500-50,000 ng of leptin in db/db mice could accelerate the removal of lipid by activating LSR.

Based on these observations, it is likely that 1) the reduced LSR activity, caused by the constantly high levels of circulating leptin, and 2) the lack of increase in plasma leptin levels during the postprandial stage, contribute to elevated postprandial plasma TG levels in db/db. It WO 01/21647 PCT/IBOO/01470 107 should be noted that the dose of leptin regulating postprandial lipemnia in ob/ob is 500 fold lower than those typically used to reduce food intake In db/db mice, leptin doses 10 fold greater than those used in ob/ob mice were needed to achieve maximal regulation of postprandial lipemia. Thus, the regulation of postprandial lipemnia in db/db mice appears partially leptinresistant, despite the fact that leptin signaling effect occurs independently of the Qb-R.

REFERENCES

Aalto-Setala, Fisher, Chen, Chajek-Shaul, Hayek, Zechner, Walsh, A., Ramakrishnan, Ginsberg, and Breslow, J.L. (1992). J Clin Invest. 90, 1889- 1900.

Alecev and Yoon (Nature Biotech. 16 :1343-1346 (1998).

Beisiegel, Weber, lhrke, Herz, and Stanley, K.K. (1989). Nature 341, 162-164.

Bihain, and Yen, F.T. (1992). Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 31, 4628-A636.

Bihain, Deckelbaum, Yen,F.T., Gleeson, Carpentier, Witte, L.D. (1989) J. Biol. Chem. 264, 17316-1732 1.

Bihain, and Yen, F.T. (1998). Curr .Opin. Lipidol. 9,221-224. Bilheimer, D.W., Eisenberg, and Levy, R.I. (1972). Biochim. Biophys. Acta 260, 212-221.

Bilheimer, Eisenberg, and Levy, R.I. (1972). The metabolism of very low density lipoprotein proteins. 1. Preliminary in vitro and in vivo observations. Biochim. Biophys.

Acta 260, 212-221.

Breslow, J.L. (1985). Adv Exp Med Biol 183,121-124.

Brown, M.S. and Goldstein, J.L. (1986). Science 232, 34-47.

Bruce, Chouinard, R.A. Jr., and Tall, A.R. (1998). Annu. Rev. Nutr. 18,297-3 Canipfield, Smith, Guisez, Devos, R. and Burn, P. (1995). Science 269, 546-549.

Chai H. et al. (1993), Biotechnol. App!. Biochem. 18:259-273.

Charron, Katz, and Olson, A.L. (1999). J. Biol. Chem. 274, 3253-3256.

Chen et al., 1987, Mol. Cell. Biol., 7 2745-2752.

Cherif, Julier, Delattre, Derrd, Lathrop, and Berger, R. (1990). Proc. Natl.

Acad. Sci. USA 87, 6639-6643.

Cole-Strauss et al. (Science 273 :1386-1389 (1996).

Cooper, A.D. (1997). J Lipid Res. 38,2173-2192.

Costet, Legendre, More, Edgar, Galtier, and Pineau, T. (1998). J. Biol. Chem.

273, 29577-29585.

Dehouck, et al J Neurochem 54:1798-801, 1990.

WO 01/21647 PCT/IBOO/01470 108 Everhart, Lombardero, Lake, JR., Wiesner, Zetterman, and Hoothagle, J.H.

(1998). Liver Transpi. Surg. 4,285-296.

Feeman, Jr. (1998) Ann. Intern. Med. 128, 73-74.

Friedman, JM (2000) Nature 404:632-634).

Friedman, and Halaas, J.L. (1998). Nature 395, 763-770.

Fuller S. A. et al. (1996) Immunology in Current Protocols in Molecular Biology, Ausubel et al., Eds, John Wiley Sons, Inc., USA Ghosh and Baccbawat, 1991, Targeting of liposomes to hepatocytes, IN Liver Diseases, Targeted diagnosis and therapy using specific receptors and ligands. Wu et al. Eds., Marcel Dekeker, New York, pp. 87-104.

Ginsberg,, Le, Goldberg, Gibson, Rubinstein, Wang-Iverson, P., Norumn, and Brown, W.V. (1986). J. Clin. Invest. 78, 1287-1295.

Goldberg, I.J. (1996). J. Lipid Res. 37, 693-707.

Goldstein, Basu, Brown, M.S. (1983). 98, 241-260.

Goldstein, Hobbs, and Brown, M.S. (1995). In The Metabolic and Molecular Basis of iherited Disease, vol. El, Scriver, Beaudet, Sly, and Valle, eds.

(New York, NY: McGraw-Hill, Inc), pp. 1981-2030.

Gopal, 1985, Mo]. Cell. Biol., 5: 1188-1190 Graham et al. (1973), Virology. 52:456-457.

Guven, EI-Bershawi, Sonrnenberg, Wilson, Hoffmann, Krakowar, G.R., and Kissebab, A.H. (1999). Diabetes 48,347-352.

Halaas, Gajiwala, Maffei, Cohen, Chait, Rabinowitz, Lallone, R.L., Burley, and Friedman, J.M 1995. Science 269, 543 -546.

Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM; Physiological response to long-term peripheral and central leptin infusion in lean and obese mice.

Proc.Natl.Acad.Sci. USA, 94, 8878-8883, 1997 Halaas JL, et al., Proc.Natl.Acad.Sci. USA, 94, 8878-8883, 1997.

Hlavel, P.J. (1998). Am. J. Clin. Nutr. 67, 355-356.

Havel, Eder, H.A. and Bragdon, J.H. (1955). J. Clin. Invest. 34,1345-1353.

Havel, and Kane, J.P. (1995). In The Metabolic and Molecular Basis of Inherited Disease, vol. II, Scrivar, Beaudat, Sly, and Valle, eds. (New York, NY: McGraw-Hill, Inc), pp. 1841-185 1.

Huygen et al. (1996) Nature Medicine. 2(8)-893-898.

Huettinger Retzek, Eder, M. and Goldenberg, H. (1988). Clin. Biochem. 21,87-92.

WO 01/21647 PCT/11100/01470 109 Igel M, Becker W, Herberg L, Jost HG; Hyperleptinemia, Leptin Resistance, and Polymorphic Leptin Receptor in the New Zealand Obese Mouse. Endocrinology, 138, 4234-4239, 1997.

Ito, Azrolan, O'Connell, Walsh, Breslow, J.L. (1990). Science 249, 790-793.

Iverius and Brunzell, J.D. (1985). Am. J. Physiol. 249,E107-EI 14 Johnson, Gallagher-Lepak, Zhu, Porth, Kelber, Roza, and Adams, M.B. (1993). Transplantation 56,822-827.

Jang et aIJ. Virol. 62 :2636-2643 (1988).

Jong, Hofker, and Havekes, L.M. (1999). Arterioscler. Thromb. Vasc. Biol. 19, 472-484.

Kandror, Stephens, and Pilch, P.F. (1995). J. Cell Biol. 129, 999-1006.

Karpe, de Faire, Mercuri, Bond, Hellenius, and Hamstcn, A. (1998) Atherosclerosis 141, 307-314 Kersten, Seydoux, Peters, Gonzalez, Desvergne, and Wahli, W. (1999). J.

Clin. Invest. 103, 1489-1498.

Klein et al. (1987) Nature. 327:70-73.

Komaromy, Schotz, M.C. (1987). Proc. Nat]. Acad. Sci. USA 84,1526-1529.

Kopelman et al. (2000) Nature 404:635-643.

Lenhard T. et al. (1996), Gene. 169:187-190.

Levit", Liu, Nouni, Meyers, Brandriff, and Mohrenweiser, H.M. (1995).

Cytogenet. Cell Genet. 69, 211-214.

Lewis, O'Meara, Soltys, Blackman, Iverius, Druetzler, Getz, G. and Polonsky, K.S. (1990)1J Clin. Endocrinol. Metab. 71, 1041-105 0.

Li, loffe, Fidahusein, Connolly, and Friedman, J.M. (1998). J. Biol. Chem. 273, 10078-10082.

Liu Q, Segal DJ, Ghiara JB, Barbas CF P~ Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. Proc Nat] Acad Sci U S A 1997 May 27;94(1 1):5525-30.

Mann, C. Khallou, Chevreuil, Troussard, Guermani, Launay, Delplanque, Yen, and Bihain, B.E. (1995). Biochemistry 34,10421-10431.

Mann, Troussard, Yen, Hannouche, Najib, Fruchart, Lotteau, V., Andr6, and Bihain, B.E. (1997). J. Biol. Chem. 272, 31348-31354.

Markwell, Haas, Rolbert, N.E. and Bieber, L.L. (198 Methods Enzymol.

72,296-3 0 Massie B, Couture F, Lamoureux L, Mosser DD, Guilbault C, Jolicoeur P, Belanger F, WO 01/21647 PCT/IBOO/01470 110 Langelier Y Inducible overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette. J Virol 1998 Mar;72(3):2289- 96.

Pelleymounter, Cullen, Baker, Hecht, Winters, Boone, and Collins, F. (1995) Science 269, 540-543.

Pengue G, Calabro V, Bartoli PC, Pagliuca A, Lania L Repression of transcriptional activity at a distance by the evolutionarily conserved KRAB domain present in a subfamily of zinc finger proteins. Nucleic Acids Res 1994 Aug 1 1;22(15):2908-14.

Perusse, Chagnon, Weisnagel, and Bouchard, C. (1999). Obes. Res. 7,111-29.

Picard, Richard, Huang, Q, and Deshaies, Y. (1998). Int. J. Obes. Relat. Metab. Disord.

22, 1088-1095.

Rohlmann Gotthardt, Hammer, and Herz, J. (1998). J Guin Invest 101, 689-695.

Romana, Tachdjian, Druart, Cohen, Berger, and Cherif D. (1993). Eur. J.

Hum. Genet. 1, 245-25 1.

Roth J.A. et al. (1996), Nature Medicine. 2(9):985-991.

Sambrook, Fritsch, and T. Maniatis. (1989), Molecular Cloning: A Laboratory Manual.

2ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Seip et al Acta. Pediatr Supp. 413 :2-28 (1996).

Shimabukuro, Koyama, Chen, Wang, Trieu, Lee, Newgard, and Unger, R.H. (1997). Proc. Nat]. Acad. Sci. USA 94,4637-4641.

Shimomura et al. Genes Dev. 12 :3182-3194 (1998) Shimomura et al.Nature 401 :73-76 (1999).

Silver, Jiang, and Tall, A.R. (1999). J. Biol. Chem. 274, 4140-4146.

Sinha, Opentanova, Ohannesian, Kolaczynski, Heiman, Hale, J., Becker, Bowsher, Stephens, and Caro, J.F. (1996). J Clin. !nvevt.

98,1277-1282.

Smith et al. (1983) Mo!. Cell. Biol. 3:2156-2165.

Snyder et (Nature Medicine 5 :64-69 (1999).

Strickland, Ashcom, Williams, Burgess, Migliorini, and Argraves, W.S.

(1990). J. Biol. Chem. 265, 17401-17404.

Tacson et al. (1996) Nature Medicine. 2(8):888-892.

Tartaglia, L.A. (1997). J. Biol. Chem. 272,6093-6096.

Teramoto et J. Virol. 72 :8904-89 12 (1998) Tur-Kaspa et al. (1986), Mo!. Cell. Biol. 6:716-718.

Uotani, Bjorbmrk, Tomoe, and Flier, J.S. (1999). Diabetes 48, 279-286.

WO 01/21647 PCTIFBOO/01470 Van Heek, Mullins, Wirth, Graziano, Fawzi, Compton, France, Hoos, Casale, Sybertz, Strader, and Davis, Jr. (1996).

Harm. Metabi. Res. 28, 635-658.

Van Heck M, Compton DS, France CF, Tedesco RP, Fawzi AR, Graziano MP, Sybertz EJ, Strader CD, Davis HR; Diet-induced obese mice develop peripheral, but not central, resistance to leptin. J.Clin.Tnvest., 99, 385-390, 1997.

Vansant, Mertens, and Muls, E. 1999) Intl. J Obesity 23, 14-2 1.

Virkami~ki, Ueki, and Kahn, C.R. (1999). J. Clin. Invest. 103,931-943.

Viasak R. et at. (1983), Eur. J Biochemn. 135:123-126.

Wang, and Sul, H.S. (1997). J. Biol. Chem. 272, 26367-26374..

Wang, Chinookoswong, Scully, Qi, and Shi, Z.Q. (1999). Endocrinology 140, 2117-21124.

Ware, C. Sanser, and Alison, E. (1998). In The Cytokine Handbook, Thomson, ed.

(San Diego, California: Academic Press), pp. 549-592.

Weigle, Duell, Connor, Steiner, Soules, and Kuijper, J.L. (1997).

Clin. Endocrinol. Metab. 82,561-565.

Wilinow, Sheng, Ishibashi, and Herz, J. (1994). Science 264, 147 1-1474.

Wong et al., 1980, Gene, 10 87-94.

Wu et al. Eds., Marcel Dekeker, New York, pp. 87-104.

Xiao et al. Virology 72:2224-2232 (1998).

Yen, Mann, Guermnani, Hannouche, Hubert, Hornick, Bordeau, Agnani, and Bihain, B.E. (1994). Biochemistry 33, 1172-1180.

Yen Masson Clossais-Besnard Andre Grosset Bougueleret Dumas J.B., Guerassimenko, and Bihain B3.E. (1999). J Biol Chem 274, 13390-13398.

Zhang, Proenca, Maffei, Barone, Leopold, and Friedman, J.M. (1994). Nature 372,425-432.

EDITORIAL NOTE APPLICATION NUMBER 76596/00 The following Sequence Listing pages 1 to 73 are part of the description. The claims pages follow on pages 112 to 114.

WO 01/21647 WO 0121647PCT/IBOO/01470 SEQUENCE LISTING <110> Genset <120> Methods of Screening for Compounds that Modulate the LSR-Leptin Interaction and Their Use in the Prevention and Treatment of obesity-Related Diseases <130> 70.WO1 <150> 60/155,506 <151> 1999-09-22 <160> 106 <170> Patent.pm <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> e223> <220> <221> <222> <223> 1 23187

DNA

Homo sapiens exon 2001. .2356 exonl exon 3540. .3884 exon2 exon 12163 exon3 exon 15144 exon4 .12282 .15200 exon 15765. .15911 exon 19579. .19752 exon6 exon 19899. .19958 exon7 exon 20056. .20187 exon8 WO 01/21647 PCT/IB00/01470 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> exon 20329..20957 exon9 exon 21047..21187 exonl0 polyA_signal 21168..21173

AATAAA

misc feature 1..2000 potential 5'regulatory region misc feature 22324..23187 homology with USF2 gene in ref: primer_bind 523..544 upstream amplification primer 17-2 primer_bind 1047..1068 downstream amplification primer 17-2 embl Y07661 complement primer_bind 946..963 upstream amplification primer 99-4576 primer_bind 1385..1402 downstream amplification primer 99-4576 complement primer_bind 1096..1115 upstream amplification primer 9-19 primer_bind 1616..1635 downstream amplification primer 9-19 complement primer_bind 1602..1621 upstream amplification primer 9-20 primer_bind 2074..2093 WO 01/21647 3 <223> downstream amplification primer 9-20 complement <220> <221> primer_bind <222> 2036..2053 <223> upstream amplification primer 99-4557 <220> <221> primer_bind <222> 2563..2580 <223> downstream amplification primer 99-4557 complement <220> <221> primer_bind <222> 2084..2102 <223> upstream amplification primer 9-1 <220> <221> primer_bind <222> 2483..2500 <223> downstream amplification primer 9-1 complement <220> <221> primer_bind <222> 2470..2489 <223> upstream amplification primer 9-21 complement <220> <221> primer_bind <222> 2062..2081 <223> downstream amplification primer 9-21 <220> <221> primer_bind <222> 3455..3474 <223> upstream amplification primer 9-3 <220> <221> primer_bind <222> 3882..3901 <223> downstream amplification primer 9-3 complement <220> <221> primer_bind <222> 3775..3792 <223> upstream amplification primer 99-4558 <220> <221> primer_bind <222> 4336..4356 <223> downstream amplification primer 99-4558 complement <220> <221> primer_bind <222> 4902..4920 <223> upstream amplification primer 99-14419 complement <220> <221> primer_bind <222> 4444..4463 <223> downstream amplification primer 99-14419 PCT/IB00/01470 <220> WO 01/21647 PCT/IB00/01470 <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> primer_bind 6638..6655 upstream amplification primer 99-4577 primer_bind 7072..7089 downstream amplification primer 99-4577 primer_bind 7995..8012 upstream amplification primer 99-4559 complement primer_bind 8576..8593 downstream amplification primer 99-4559 primer_bind 9622..9639 upstream amplification primer 99-3148 complement primer_bind 10023..10040 downstream amplification primer 99-3148 primer_bind 9964..9981 upstream amplification primer 99-4560 complement primer_bind 10546..10563 downstream amplification primer 99-4560 complement primer_bind 10996..11015 upstream amplification primer 99-14411 complement primer_bind 10492..10512 downstream amplification primer 99-14411 primer_bind 11972..11990 upstream amplification primer 99-4561 primer_bind 12481..12501 downstream amplification primer 99-4561 complement primer_bind 12005..12023 upstream amplification primer 9-4 WO 01/21647 PCT/IBOO/01470 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> primerbind 12417..12436 downstream amplification primer 9-4 primerbind 14102..14119 upstream amplification primer 99-4562 complement primer_bind 14543..14563 downstream amplification primer 99-4562 primer_bind 14431..14448 upstream amplification primer 99-3149 primer_bind 14848..14865 downstream amplification primer 99-3149 primer_bind 14748..14767 upstream amplification primer 9-22 complement complement primer_bind 15198..15218 downstream amplification primer 9-22 primer_bind 14748..14767 upstream amplification primer 9-24 primer_bind 15333..15351 complement downstream amplification primer 9-24 complement primer_bind 15002..15019 upstream amplification primer primer_bind 15333..15351 downstream amplification primer 9-5 complement primer_bind 15640..15657 upstream amplification primer 9-6 primerbind WO 01/21647 6 <222> 16072..16089 <223> downstream amplification primer 9-6 complement PCT/IB00/01470 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> primer_bind 15800..15817 upstream amplification primer 99-4563 primer_bind 16179..16199 downstream amplification primer 99-4563 primer_bind 19295..19312 upstream amplification primer 99-3150 primer_bind 19729..19746 downstream amplification primer 99-3150 primer_bind 19420..19438 upstream amplification primer 9-7 complement complement primer_bind 19824..19841 downstream amplification primer 9-7 primer_bind 19798..19815 upstream amplification primer 9-8 primer_bind 20137..20155 downstream amplification primer 9-8 primer_bind 19913..19931 upstream amplification primer 9-9 complement complement primer_bind 20329..20346 downstream amplification primer 9-9 primer_bind 20139..20157 upstream amplification primer 99-4564 primer_bind 20582..20599 downstream amplification primer 99-45 complement 64 complement WO 01/21647 PCT/IB00/01470 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> 7 primer_bind 20238..20256 upstream amplification primer 9-10 primer_bind 20645..20662 downstream amplification primer 9-10 primer_bind 20410..20424 upstream amplification primer 9-26 primer_bind 20690..20706 downstream amplification primer 9-26 primer_bind 20569..20588 upstream amplification primer 9-23 primer_bind 21243..21262 downstream amplification primer 9-23 primer_bind 20583..20604 upstream amplification primer 9-11 primerbind 21015..21034 downstream amplification primer 9-11 complement I complement complement complement primer_bind 20584..20601 upstream amplification primer 99-15285 primer_bind 20139..20158 downstream amplification primer 99-15285 primer_bind 20642..20659 upstream amplification primer 99-15287 primer_bind 20207..20227 downstream amplification primer 99-15287 complement complement primer_bind 20691..20709 WO 01/21647 8 <223> upstream amplification primer 99-15286 complement PCT/IBOO/01470 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> primer_bind 20238..20257 downstream amplification primer 99-15286 primer_bind 20943..20960 upstream amplification primer 9-2 primer_bind 21295..21312 downstream amplification primer 9-2 complement primer_bind 21013..21031 upstream amplification primer 99-15284 primer_bind 20582..20602 downstream amplification primer 99-15284 primer_bind 21019..21038 upstream amplification primer 99-14407 primer_bind 20571..20589 downstream amplification primer 99-14407 primerbind 21079..21097 upstream amplification primer 99-15283 primer_bind 20638..20655 downstream amplification primer 99-15283 primer_bind 21013..21032 complement complement complement upstream amplification primer primer bind 21195..21214 downstream amplification primer LSRilOrl4s complement primer_bind 20354..20372 upstream amplification primer LSRx9fl3s WO 01/21647 WO 0121647PCTIIBOO/01470 <:221> <222> <:223> <220> <221> <222> <223> <220> <221> <:222> <:223> <220> <221> <222> <223> <220> <221> <222> <223> <:220> <221> <:222> <223> <220> <221> <222> <223> <220> <:221> <222> <223> <220> <221> <222> <:223> <220> <:221> <22 2> <223> <220> <221> <:222> <223> primer-bind 20570. .20591 upstream amplification primer LSRx9fl4s primer Tbind 20811. .20832 downstream amplification allele 818 17-2-2 97 allele 1243 9-19-148 allele 1374 9-19-256 allele 1401 9-19-307 allele 1535 9-19-442 allele 1788 9-20-187 allele 2391 9-1- 308 allele 3778 9-3-324 polymorphic *polymorphic polymorphic polymorphic *polymorphic polymorphic polymorphic polymorphic primer LSRx9rl3s, complement base G or C base C or T base A or G base A or T base deletion of C base A or C base G or C base C or T allele 4498 99-14419-424 <220> <:221> allele <:222> 15007 <223> 9-24-260 :polymorphic base T polymorphic base A or or G

G

<220> <:221> <222> <223> allele 15233 9-24-4 86 polymorphic base A or G WO 01/21647 PCT/IBOO/01470 <220> <221> allele <222> 15826 <223> 9-6-187 <220> <221> allele <222> 19567 <223> 9-7-148 <220> <221> allele <222> 19744 <223> 9-7-325 <220> <221> allele <222> 19786 <223> 9-7-367 <220> <221> allele <222> 20158 <223> 9-9-246 <220> <221> allele <222> 20595 <223> LSRX9-BM <220> <221> allele <222> 21108 <223> LSRX10-BM <220> <221> allele <222> 606 <223> potential <220> <221> allele <222> 5141 <223> potential <220> <221> allele <222> 7428 <223> potential <220> <221> allele <222> 8394 <223> potential <220> <221> allele <222> 8704 <223> potential polymorphic polymorphic polymorphic polymorphic polymorphic base C or T base A or G base A or G base A or C base G or C (17-1-240) polymorphic base deletion of AGG polymorphic polymorphic b.

polymorphic b polymorphic b polymorphic b polymorphic b base T or G ase C or T ase insertion of G ase insertion of C ase C or G ase T or C <220> <221> allele WO 01/21647 PCT/IB00/01470 <222> 9028 <223> potential <220> <221> allele <222> 9950 <223> potential <220> <221> allele <222> 9977 <223> potential <220> <221> allele <222> 10021 <223> potential <220> <221> allele <222> 11878 <223> potential <220> <221> allele <222> 19040 <223> potential <220> <221> allele <222> 21363 <223> potential <220> <221> allele <222> 21449 <223> potential <220> <221> allele <222> 21451 <223> potential <220> <221> allele <222> 21454 <223> potential <220> <221> allele <222> 21455 <223> potential <220> <221> allele <222> 21569 <223> potential <220> <221> allele <222> 21683 <223> potential polymorphic polymorphic polymorphic polymorphic polymorphic polymorphic polymorphic polymorphic polymorphic polymorphic polymorphic polymorphic base G or A base deletion of GAATGAAA base T or C base A or G base C or T base deletion of G base A or G base C or T base G or C base A or G base G or A base T or A polymorphic base deletion of C WO 01121647 WO 0121647PCT/BOO/O 1470 <220> <221> allele <222> 21694 <223> potential <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> polymorphic polymorphic base insertion of T base deletion of G allele 21728 potential misc -binding 799. .817 17-2-297.niisl rrisc -binding 819. .837 complement 17-2-297.mis2 misc -binding 1224. .1242 9-19-148 .mi 61 misc -binding 1244. .1262 complement 9-19-148.mis2 misc -binding 1330. .1373 9-19-256 .misl misc -binding 1375. .1393 complement 9-19-256.mis2 misc -binding 1382. .1400 9-19-307 .misl misc -binding 1402. .1420 complement 9-19-307.mis2 misc -binding 1516. .1534 9-19-442 .mis 1 misc -binding 1769. .1787 9-20-187 .misl misc -binding 1789. .1807 WO 01/21647 PCT/IBOO/01470 13 <223> complement 9-20-187.mis2 <220> <221> misc_binding <222> 2372. .2390 <223> 9-l-30B.MiS1 <220> <221> misc -binding <222> 2392. .2410 <223> complement 9-1-308.mis2 <220> <221> misc -binding <222> 3759. .3777 <223> 9-3-324.misl <220> <221> misc -binding <222> 3779. .3797 <223> complement 9-3-324.mis2 <220> <221> misc -binding <222> 4979. .4997 <223> 99-14419-424.mis2 <220> <221> misc binding <222> 4999. .5017 <223> complement 99-14419-424.misl <220> <221> misc -binding <222> 14988. .15006 <223> 9-24-260.misl <220> <221> misc -binding <222> 15008. .15026 <223> complement 9-24-260.mis2 <220> <221> misc -binding <222> 15214. .15232 <223> 9-24-486.misl <220> <221> misc -binding <222> 15234. .15252 <223> complement 9-24-486.mis2 <220> <221> misc-binding <222> 15807. .15825 <223> 9-6-l87.mial <220> <221> misc -binding <222> 15827. .15845 <223> complement 9-6-187.mis2 <220> WO 01/21647 14 4221> misc binding <222> 19548.-.19566 <223> 9-7-148.misl <220> <221> misc binding <222> 19568. .19586 <223> complement 9-7-148.mis2 <220> <221> misc -binding <222> 19725. .19743 <223> 9-7-325.misl <220> <221> misc -binding <222> 19745. .19763 <223> complement 9-7-325.mis2 <220> <221> misc binding <222> 19767. .19785 <223> 9-7-367.misl <220> <221> misc -binding <222> 19787. .19805 <223> complement 9-7-367.mis2 <220> <221> misc -binding <222> 20139. .20157 <223> 9-9-246.misl <220> <221> misc -binding <222> 20159. .20177 <223> complement 9-9-246.mis2 <220> <221> misc binding <222> 20576. .20594 <223> LSRX9-BM.misl(17-1-240) <220> <221> misc -binding <222> 20596. .20614 <223> complement LSRX9-BM.mis2(17-1-240) <220> <221> misc -binding <222> 21089. .21107 <223> LSRX1O-BM.misl <220> <221> misc -binding <222> 21109. .21127 <223> complement LSRX1O-BM.mis2 <220> <221> misc -binding <222> 587. .605 <223> potentialsite6O6.inisl potential PCT/IBOO/0 1470 WO 01/2 1647 is <220> <221> misc binding <222> 607. .625 <223> complement potentialsite6O6.mis2 potential <220> <221> misc binding <222> 5122. .5140 <223> potentialsite5l4l.misl potential <220> <221> misc-binding <222> 5142.. 5160 <223> complement potentialsite5l4l.mis2 potential <220> <221> misc -binding <222> 7409. .7427 <223> potentialsite7428.misl potential <220> <221> misc -binding <222> 7429. .7447 <223> complement potentialsite7428.mis2 potential <220> <221> misc -binding <222> 8375. .8393 <223> potentialsitea394 .misl potential <220> <221> misc -binding <222> 8395. .8413 <223> complement potentialsite8394.mis2 potential <220> <221> misc -binding <222> 8685. .8703 <223> potentialsite8704 .mial potential <220> <221> misc -binding <222> 8705.. 8723 <223> complement potentialsite8704.mis2 potential <220> <221> misc -binding <222> 9009. .9027 <223> potentialsite9028 .misl potential <220> <221> misc binding <222> 902g^.."04"7 <223> complement potentialsite9O28.mis2 potential <220> <221> misc -binding <222> 9931. .9949 <223> potentialsite9950.misl potential <220> <221> misc binding PCT/IBOO/01470 WO 01/21647 PCT/IBOO/01470 16 <222> 9951.-.9969 <223> complement potentialsite9950.mis2 potential <220> <221> misc -binding <222> 9958. .9976 <223> potentialsite9977.misl potential <220> <221> misc -binding <222> 9978. .9996 <223> complement potentialsite9977.mis2 potential <220> <221> misc -binding <222> 10002.-10020 <223> potentialsitelO021.misl potential <220> <221> misc -binding <222> 10022. .10040 <223> complement potentialsitel0021.mis2 potential <220> <221> misc -binding <222> 11859. .11877 <223> potentialsitella7a.MiSl potential <220> <221> misc -binding <222> 11879. .11897 <223> complement potentialsitell878.mis2 potential <220> <221> misc -binding <222> 19021. .19039 <223> potentialsitel9040.misl potential <220> <221> misc -binding <222> 19041. .19059 <223> complement potentialsite19040.mis2 potential <220> <221> misc -binding <222> 21344. .21362 <223> potentialsite2l363.misl potential <220> <221> misc -binding <222> 21364. .21382 <223> complement potentialsite2l363.mis2 potential <220> <221> misc -binding <222> 21430. .21448 <223> potentialsite2l449.mial potential <220> <221> misc -binding <222> 21450. .21468 <223> complement potentialsite2l449.mis2 potential WO 01/21647 17 <220> <221> misc binding <222> 21432. .21450 <223> potentialsite2l45l .misl potential <220> <221> misc -binding <222> 21452. .21470 <223> complement potentialsite2l45l.mis2 potential <220> <221> misc -binding <222> 21435. .21453 <223> potentialsite2l454 .misl potential <220> <221> misc -binding <222> 21455. .21473 <223> complement potentialsite2l454 .mia2 potential <220> <221> misc -binding <222> 21436. .21454 <223> potentialsite2l45s .misl potential <220> <221> misc -binding <222> 21456. .21474 <223> complement potentialsite2l455.mis2 potential <220> <221> misc -binding <222> 21550. .21568 <223> potentialsite2l569.misl potential <220> <221> misc binding <222> 21570. .21588 <223> complement potentialsite2l569.mis2 potential <220> <221> misc -binding <222> 21664. .21682 <223> potentialsite2l683 .mial potential <220> <221> misc -binding <222> 21684. .21702 <223> complement potentialsite2l683.mis2 potential <220> <221> misc -binding <222> 21675. .21693 <223> potentialsite2l694 .misl potential <220> <221> misc -binding <222> 21695. .21713 <223> complement potentialsite2l694.mis2 potential <220> <221> misc binding <222> 21709. .21727 PCTIBOO/01470 WO 01/21647 18 <223> potentialsite2l72a .misl potential <220> <221> misc-binding <222> 21729.. 21747 <223> complement potentialsite2l728.mis2 potential <220> <221> misc-feature <222> 22113,22122,22227,22264,22268 <223> n=a, g, c or t PCT/IBO0101470 <400> 1 ccataatcaa gtgcattgct ctcaaaaggt tcaaaagaaa gtcaaaaggt tatacataca catggcttcg actagacaca ttggtaagca ctgtt taatg ggtgactggt gttaaagcga gaagggacgt ggtgtcctac ggagtagcac gccagggtgt aaggtcagtt gatctggctt cc act ggcc a ggatgcgctt ctgatcgccg gcggaaaccg gagggctgac ggaggtggcg gcatccctga aactgggaga tggaagtgga tcgctcctcc ggcagggagg agtgggtctc gaagggacct ggggacggag cgccgctcac tcaccggagc tctcccccat agaa tgc aa c gtgcacccct cgggccagac ctgggctccc agcacctggt gctgggacct ttccccattt aactccctgg gtggaagac c ggat ttc taa tagagggaca ccccatcccc tggtgggatg ga tat ttggg gaaaatggat ggtgggaatg taaaaataga tgaaagcaga agaaacaacc atggtacact acatactaca ggacaaatgt aagtagaata gtcacagagt acacgacatt taaatattat gaacggg ttg aggaggagga gggcgatggc ccgaagctga tctgctgaga gagttttaac gt tggcaacc tccctcgccg cgccgcgccc g tcc caagcc ccggggctag aaaacttccg gggggcagag cacaggaggc gaagtcaggc cggcagggcg gaacaggtgc ggttgcatgg gtggtccgca cgcacctggg ccctaagccc gcgcagcggg atctgaaagc aggacggact cctggccttg gcgcccagac acccggccgc gcacaggtac ggatggatgg tccgatcccc gtgccgggga acggtgagac gcccgatctc gttttggagt gagacgaagg cgcaccccac gatttacgga tggtgatgag aataagtttt tacaatagtg actaccaagt tatttgtaca taggtccatc agtccgctgt acatggatgg tagacgattc gaaattacta ttatgttggg gtgaatatac agtttgcata aaattttata agatcatgtg tctgaggact gtaagccaga gtgaggagga agggccagta tgggggagtc c cacct ggag ccgccctcgg ccgcgccttc ggatgcccag wagtggaatt cttcccttcc ccgaagcgtc gccaccaaca cacctaggcg gcggcgggac atctcggtgc ggcgccctcC tcagcccact agccctcggc tggagtgtgg atgccctttg tggagt aggg gtgcgcgccg ggccgcgatq cgcaggccgg ggggcaCggg agttggaggc tgggtcttgg gcgctccccg ccggagcggc tgggaaaaga aacggggaga aacaatgcct ccacctcccg cttgaaaccg gtctttcccg ggtggggatg cagtcattgg cacccagcaa ccagtgttca cacaaatgaa aaaaagaaat accttgaaaa cacttatatg ggggcacagg gatgatgaaa ttaatgccac ttttatcata aaaagtggcc gacatctscg gtgagaagta gagagtggga gccacaggag gaacaaagca taacgcgagg acagcagagt tcgcaggtgg gctcggcccc gggccgaacc ccaacttttc ggggacttta ccccctccac agcctctccc ggtccatcgc ccgccctatc gcttggtttg cagcgggcca tccggggagg tcccaccctt ctcggaggac tccacgtcgt acaaggaacg cgccccctaa gcgctgt tgg gacgcggtcg gcctctgacg ggcgggaagc gcgatctgtt cgcccttatc agggagaatg aaagccggga gggcacggga aggcctcggg cgggctccgg acttctgctg agacactttt tggagaaatt ggaaaacagt ttccattctt cagctgcact tggataaata tttgatctta cattattctt aggcacctag tagcagggaa cagtttcggg tgaattttac aaaatatttt agggaaggtg ggaagggtgt tagt tggaaa ggaggtgaga ggctgtgagc cgcctgggta aagcgcccag cacgcccagc ctygttccgg tttaagagcc acaagttggg ctggccctga gagggttcct ccggtccgga agccaggact cagccgggga tcaacaggtg gccggagnag gtcacttggt gaggcagagg gtgtacctgg cgcggcgggt ttacgctcat gaagtgggaa ggtactttgg ccggcgggct tcttcgtgtg c tgcggaacg gggaagC9gg gcgcgcggga tggaagatag gaactctttg tgggacttgg gggctggatc tgggtgtggg attatacgtg gcaggctttc ggttcagtca ggaatcctcc ttggcagttc aggcatatat atttacaata aaacgtagca c tgc atgc ta tgtgaaataa aatgggcaat tggggagtta gataaagagt acttgaagtg tttaaacgat tcactgcaat tctggcagag cagcgaggag taagaggggg aggtggacgt ccgaaaccag ggttccccca gctgcgcagg gaattcctaa agaatttccg aacrggtggg ttccccttgg caggtcatct ggaaccccag ttgcttagac gaggggtttg aatcgctcca atgggggccg tcgggccctg aacccctccc gccgaaccat caagcacctt taaaacttcc ggggaggagc aagggacgcg ctccagaggg gcttctgctt scggagggaa ggtctcagag gtgagaggaa caggaagtga tggggaggga gcgcacccgg agacgcttcc actggggact cgtaagagtc acctggatgg tttgaaatga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 WO 01/21647 WO 0121647PCT/IBOO/0 1470 ctttagagta ccatggcaaa cgggtaacct caacatggcc tggcaaaggg gggcgagtcc tataaaatgg gtgccaggca ctgttgaagc tcctgccagg gcctgtgacc ctggaagtac cgacaaccag gtgccaggac cctgggagat agggggatga gtccccatct tttttttttt tgcgatctca ctcccgagta agtagagatg tctgcccgcc ttctattcta tttttgagta aaaatcatca acacactctc ttgttctata tataaagggt cacagtaatt cttgcctagt gtggccaagc gatggcagga ggaactgcac cagagaggtt aacccaggca ccaggctgga gccattctcc ggctaatttt gatctcctga gagccaccgc tgttgggact ctatgtgtcg ggagatgaca agaatttcaa aggtgggtgg ccgtctctac ctactcagga ctgagatcgc aaaaaaaaaa agaaaataac gccaaggcag gaccccatgt ctgtggtccc ggcttcagtg gtcttgaaat ttgggaggcc atggtgaaat tgtaatccca gttgcggtgc ttaaaaaaaa ctgtgaatat ggaaggggag t tcactgtct gggtgaggtg gcagttcagg ggaatccttc atctgggctt aagaggggac a aggaaa ctg gcatgattat ctctgtaaac acctccctga gccatccagg ctgccctgta aagtctttct ctcaatgccc agcgtgcgca tactaccagg ggctgggctt gaaagctctt tttttttgaa gctcactgca gctgggactc gggtttcacc ttggcctcgc ctattcttcc cctgtcatat aaccacttcc aagtccttga atcattaata ctactgtgtg gaatagattc gtcatagctg tcctaatccc gtgaactgca acatattctc accttgttca gccctgagaa gtgcagtggc tgcctcagcc ttgtattttt cctcgtgatc gc ccggccc c aagt aagggg ttggtgctgg ttctggtgga agtc tgggca atcacctgag taaaaataca ggctgaggca accactgtac aaaagactcc accaggctgg gaggatcact ctacaaaaat ggctgctcag agtcatgatc gaaaagaaaa gaggtgggtg cccatctcta gctactcggg gccaagattg aagaaaaaag gatttgagga gggcagggtg taaacaagaa gtgggaggct caaacaactc gtagtcggtt gcctgcccag tggtgtggtt gccgtgtcac cgtggctacc cacatacttg gctcacagca tgaccgtgtc cctaccagat gccgggaccg agc tggcagc ccgtcagggt gccggaggat gcccgggtgg gagtgccagt gatggagtct acctccacct caggtgcgtg atg tt ggc ca aaagtgctga ttctgctaat accaggtgct ctcgtggagc agataaagaa acacgagcag c taag ctc tc tatcattgtc gtaacggggc tttgcttgcC ggggcaagag ccattgagtc aatgggtgca ccttgttctt gggatctcgg tcccaagtag agtagagacg cgcccgcctc ttgttcttaa cagtcattca ggatagaggt gtgagactgg cggtggctca gtcaggagtt aagattagcc tgagaatcgc tgcagtctgg gtcaaggtat gtgcattggc tgagcctagg tttaaaaatt gaggctgagg gtgccactgc taggc tgggc gatcacctga ctaaaaatac agyc tgaggc cgccactgca aaaatagcac gggcc tgggc ccagggatga tttaaagttg gatggagata catggttttc ttgaagtggg gccacacaga cagaggccac cgtgcacctg tcactggtcc cgagtgtcaa acccttgctg caacccctac gacctcgacc catcgccgat cgggaaccca cgtggccacc taccatcacc tgggactggc gtctgaaagg tgctctggac cccagg tt ca ccaccacgcc ggctggtctc gagacaccat ccttccattc gttctgggcc ccacat tgca aactgggtaa taagaagtaa atccaggttc atcttacaga tgggattcta tagggtggtc agcttaatgg ctctcctcta taggaagtca tttttttgag ctcactgcaa ctgggactac gggtttcac ggcctcccaa ctgtaatgct ttcattcatt gattgggatg cagtaaataa cgtctgtaat ccagaccagc aggcatggtg ttgaacccag gcgacagagt aagaatgtca tcatgcctgt agtttgagac taaaaattag tgggaggatt attccagcct gcagtggctc ggtcaggaga aaaatttagc aggagaatcg ctctagcctg tgggtgatgt ctgggcctta aggctcacgt ggcatggggc ttgtgggggc cctccaaatt gccttgggcg ggcccct tgt aggc tgggaa ccacttcagc tggcaattaa gc tggtgaca tc tc tccLc t cacgtggtga cccacgcaac gccttctccc ggctacaacc aagcagggca ggaagtatgt gtccttgtgc accattgaag tccaggctgg agcaattctc cagttaattt aaactcctga acccagccta tttaatttaa ctgggaatac gtgagagaga cggagagaag aatttatcta cc aaggat ta ggcccagaga actcagccac cgcagaggac agaaagcctg ccctcctgac aagtctggag acggagtctc gctccgcctc aggcgcccgc gttttagccg agtgctggga gcctcctgat tggtatttat gctgaagtt t gcagataaag ctcagcactt c tggcc aaca gcacatgcct gaggcagagg gagactctgt gagagtacta aaatttcagc cagcctggac ctgggcatgg gcttgggctt gggtgacaga acacctgtaa tcgagaccag cgggcgtggt ct Lgaacctg ggaaacagtg gctacatgga cagaacctag acctcatgtc agagcgggga tttagtccct caaaaggccc ctgggggcat tgtgggtgaa gagggatggc cccacgggtc ggaacaatgt ggtggcgttc tgccctcagc tcctcttcca ccatcgtcat cggccagcgt cctacgtyga acgctgtgac tgggcagggc gggacctgga ggagcaat tc agtgcagtgg ttgcctcagc ttgtattttt cctcaaatga aagggagcga taacgaagat agctgttaac caaacackac aggccagggt agtaacaact actcagacca gagaaagtga tttgggtcta tcacagagga tgacatgcca agctgaggca ctggggittg gctctgtcgC ccgggttcac cactacgcct ggatggtctc ttacaggcgt aggatgtgcc c aagcatcga ctgtcgtcaa aaagagtatg tgggaggcca t ggtgaaacc gtaatcccag ttgcagtgag ctcaaaaaaa agtgttgcaa actttgggag aacaaaatga tggcatgtgc gagaggtCaa gtgagaccct tcccagcact cctggccaac ggtgggcgc ggaggcgaag agactccgtc atgacttggg aaggcagaga ttagtgtgtg agggagcatc 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 WO 01/21647 cctttgcaga gcagggttoc tgccctgaat gctgcttagg gctaagcttc too agga tga acaggcctgg gtaaacatcc ttcagctttg gotggggttt caatctcagc cctgagtagc ggtagagatg tctgcctgc tatcctggtt a toCot ccgg agcacctgga acctccagct tgcggtgtat cactggagac tgagatcacg cagaccttag gtcagacagg caacccctgg ttatagcagg gaagacactq tgcttgagga aaaaaataaa cttgagccta ggcga ccaag cattgtggca aaagaatgtg gggtggtggo gagccctgga aacaacacca cctcaacccc ttgtttaaaa tttctccata ttctttccta attccgggct agcgaggtgg tgaggtcagg atacaaaaat aggcaggaga tgcactccag gctcaaagag gacaataaag tttctgaaat gtctccgaag gatatctatt taactttgac agtttgagaa gtactcagta aatgtagtott gactttagca tctcactcta cctcctgagt gtgccaccat gccgggc tgg ttaggat tac ccaggtattg cacggagccc ttcagcagtt PCT/IBOO/01470 ccccaagaag aggggctgag ttctgtgggO ccccctcctc tgccaggaca ccccccaggc gtctttcaaa attgtgtgtg aggatgggga ttctgttgtt tcactgcaac tgggattaca ggatttcgcc ttggcctccc tcaaaagtga tccggtggct caggaggcco gcactgcagc aaagaaggaa ctggcatctt gccagcccaa gatggatcaa tcaaacaaaa cctatatgat gaccagctct gtggctcatg caggagctgg tttagcgggg ggagttcgaa ctaggccctc aacctgggga tgtagagggg tcatgcctgt gttcaagatc acaacaaaaa ttcacatctg tggcacagcc cagcttatct gttagaatct taggcctctt ctcatgcctg agttccagac tagccagtta atcgcttgaa cctgggcaac tttgttttca tcattataga cagacaaaac cctggctgga gtatgtcacg ctcaggatct ctgttgctgt attattgttg tgaatctagc tgaggagagg ctgcccaggc tcaagcaatt ge t agct aa tctggaactc aggtgtgagc tgtcaggcac acattttcac gggtggggga ccaggaac tg cctatgtcag agctcagcca tctaacccag agccaaatac tctgtctcgg ccacccccac tcctttcagg aaagtggact gttgtttttt ctctgcctcg ggtgcacacc atgttggcca aaagttctgg aaatagtcct cattccctgc tgttccacct agagagccca ccagaggttg ctccccgacc gaccaggaac aagaagccgg cacagtgagg ctgtttgcca tgagctccag cctgtaatcc gagacoagc catgatggca gctgcagtga tcaaaaaaga tttaggagaa cccagcaca t aatcccagca aacctggac a aacagtggct ctctgaaggt cccactccac ttgtctggta gaactctaga ttatattctt gaatcccagc ctgcctggcc tgttggcagg cccaggaggc agagcgagac taggcagcag ttaacataaa attgtgaatg tagaagagcc tagcctctgg cctgaaaacg ggtatcccag aaggatgaat catgtgcoca gggttcgttt tagagtgcag ctcctgcctc ttttttacat ctgacctcaa caccatgtcc tggggaccca caggcaaagg gccgtgggag gagcacattc tcctgtttca tgagcatcct ggattgtgcc tgagggtgct ggaaggggcc ctgggcctgt aaatcccata coccgtggtg ttctgttgcc caagt tcaag accacacctg ggctggtctc gattacagat ggataaggta tttgtccttc cctogggoto tgggacctct gagatgtgga taaaacaatg agggtacgcc aaaactatat tccagootog tggcaggcgg tgttgaagag cagcacttca tgggcaacac caccctgcta cccatgatcg tacaggtgga cctagtttgg agtaagagct ott tgggagg acaaagcaag tagatgcctg cacoatatoc gcctggcact tgtgacatag agggcaaggg gattttgagg actttgggag aacacagtga cgcctataat agaggctgca tccatctcaa aatgagaaaa taaaatacct agaaggtggc tggcttctca aaaactccac tot tggggaa gtgtctcaaa gaatgaatga gaatcatoac cctttttttt tggtgcaatc agcctccagg ttttggtaga gtgatccacc ggccaagagg gcagtggctg atggtcggoc aagcccagcc tgctagagga gaggaggcac actgttattg tgcctggacc tcctctgctg ctgcatgctc gtttgatcaa gccataggag ttcctagggt caggctggag tgattctccc gctaattttt aaactcctga gtgagccacc gaaggctgtc catgctttgg cttggggtcc gaaatcatga ggaggcctgg aaagcagtgc ctgcaggaag tttttgtgag gcctacaaga ttcctgtcca go a gg t cag ggaggccgag agtgagaoccc ctctggagat caccactgca aaaatgatgg aattctatga caataaacgg ctgaggcagg atcccatctc atcattaggg ggaagccttc ctotgctgto ttaacatttt caaggattta ttaattaaga gcccaggcgg aaaacctgtc cccagctact gtgagccaag aaaaaaaaaa gtttacaaaa tttatgaaaa atggttttat gagctgcttc agttagtatt ccccagggtc tactgcctag atgaatgaat acacfatgAoa cttttttttt tcagctcact gtagctggga gatggggttt tgcctcagcc gtgttcattt agacagacag cctaagctgg cacaggggga tcgatgggaa caggcttgct aggtcacagg aggcgtgact gacgcaaaag caggggcctc ggccctgagt Cttcctctgt cacccactgt tgcagtggtg gcctcagcct gtatcttttt cctcaggtga atgcccggcc cactccaggc gtgatggaoc aagtgcccoc aggtcacctt otgctgttco tcagcccgga aaggtgtgcc ttttgaaaat tgccagattt cctcttttgt gg totga tot gcaggaggat agacactaca gggaagattg ctccagcctg acgaagaggg ggattoaatg tgggggctag tgga tca Ott aaaattaaaa taagtcgtgt cc tggcc tcc cctgattcgt atatttgtct taactcaaag gctcaggcct gcagatcact tctactaaaa caagaggctg atcgtgccac aaaattaaga tagtttaaat aaataatcat ttttttgcaa agtctgttgt gttgggaaaa tagaggctgc aacataggtg gaaagaaaga aaagotaagg tgagatggag gcaatctctg ctacaggtgc taccatgttg tcccaaagtg ctgctccttg ggotctgoct gagat aagac cagtgcaaat 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 WO 01/2 1647 ctagaaccaa aagggccttg cagacactgt agggttcttt cgtcacagag gctaggagga ctacttctgt ggtgaaaggc aaaacacttt agcgtcccca caaggcactg cctgggcatc ctacactagg ggtggttcag ctgtaatccc aaaaccaact ggtgtggtgg tgaatctggg ctgggcaaca gagtgcctgt tgcagtgggc tctaaagaaa tgaagagctg gtcctgcctc tgtgcccttg tgcttgcccc ccagcctcca accagacggc tccaggggaa ggggaggcat cctctgccct gc cagt cact cctccaatca caaagtc Lca taatcccagc tagcctggcc gtgcacatgc gggaggcaga gtgagactct tgaggacagt ctcacccgag tgcatggcac ccaggggcag ccatcggcca catgagtgat tggggggtac tgacccagga aaattagtct gtttgtttct agattttcct agtgcagtgg ctgcctcagc tttttgtatt tgacctcgtg tgcccggcct catggcaaga ctgtgagccc ggctgcagtg caagaaacac gatgatggca aaggcaaata cacgctttga gtgcatgagc PCT/IBOO/01470 ggcgatggca gggaccatgg tgcaaggagg cattcctggg ctgtcatgca aagaggccgg ccc cag tggc tgaaggtatt gatggatggt agccctgggc ggagggtaag tcctggtacc ccctgtgggt agatggctgt agcactttgg tggtcaacat caggtgccta aggcgaagat agagtgaaac agtcctaact tataattaca gaaaaaaaaa tgcagctttt acagacttct gtaggaatct ctcctgggtg aggtgccctc gtggggggac c aat gaggcc ggcccttcct ccagcttacc taggctcccc cccaagccaa tttttaaaaa actttgggag aacgtggcaa ctgtaatccc ggatgcagta gtccaaaaaa tgatggtgtg tccctcgcca acacactgtg ggcggggtgt ggtgccctgc gccgctgggc ttgggctgca acctctgcct ccaggtctat gtgggtttta tttttttttt ctcgatctcg ctcccaagta tttagtagag at'cacccac taaaatgggg ccctgactct agcttcttgg agccatgact aaacacacac tcagggagcc acatcttagt gagctgtgac t ctggggagg ggggtgaggc gactatggga atttttctgt ggtgccaggt ggagggcagc gaagc tgagg t taaggcaga taattagtgc agagttaaca c tgacc tgga tctctctctc tagaatctgg ggcagttcac aagt tgagaa gaggctgagg ggcgaaactc taatcccagc tgtagtgagc tccgattcaa actcaggtgg ccactgcact aacaacctca ccagacccga tgcagtagtc ttgcatatgc tgctgggaag acgccttttc agtggtgtgt tacgcagagc tttgtccgct ct ctgggct c tgtgccctgc actaagagaa aaatccagac gccgaggcgg aatcccgtct agttactcag agccaagatc aaaaaaaatc caaaatcgac aagcacagcg taagtttgag ccgtgtggct tgtaagccaa tgtacactgc agacagagtt gt tcttcggt ttacactcta tgtctatctg ttttttttga gctcactgca gctgggacta actgggtttc ct-gggcctcc agatttttca acaaaaaatt gaggctgagg atgctactgc acacacacac tgggaaactc gttattctaa tgacatgcct gcttcgtggg tggcacggta cct:tagggaa agaca tgagg gctgtggact gcgtccttgg gcatttcctg gttgcctggt ctgagaagca ggctccttgc atgtggggat ccacatagac cctgtttatc atcaggggag gcacagactg tgggggtgga catctctact tacatgggag cgagattagt acaaacaaaa gaggattgct ccagccaggg ggctccgagg aatgtcatcc caggtgttta atttgatcat gggtctttgg atcCCgactc attactgctc tcatcgtcct tctgt Lctgt tgtcgcctgc accccaggca gagtggagac ttggggtccg gtggatcact ctataaaaaa aaggc tgagg aagccactgc cagacgtggt ccacc tcttg gttttgccgt tgcagctgaa gggaggcctc cacatggagt catcttcaca catgtgtggg aaaatgcac aaacattatc ttgct taaca gatggagtct agcttcacct caggcacccg accatgt tag caaagtg'rtg agcccaagat ttaaaattaa caggagtatc actctagcat acacgcatat tactggacat aatttcttct ctgtcctgtt tgcagagctg gciagagacc ggcgtctgga ccttccttat gcagcacgcg gaaggtggca cc cgagatgc aggtgccctg gagaggaaac ctgcagctgc ggaaggggag acacccactc tccacaccca ttctgacttt ctgggtgtgg tcacctgagg aaaaatgcaa gctgaggcag tcgcaccatt aaaaaaagct tgagtccagg ccacagagtg gcaccattac acaaaacaga gaacggggtg ctgcagcctg ccctccaggg agatgctgac cgtggtctca tggtgagtgg ctgccctccc tctgctctcc gggaccactg aattggagac ggtgcggtag tgaggccagg tataaaagcc catgaggatt actccagcct cagagtccat ctacatcccc gtgcctgct acgaagccga cttgtgttag cttgtatgac tgtgtgaatg ggatggaaca atttgcatca gagggtctcc tattaggaat cgttctgtcg cctgggttca ccaccacacc ccaggatggt ggattacagg acacaaggaa ccaggcatgg gcttgcaccc gagtgacaga agtccattag tcatgggaga tttggccttg gcgagggcct ggcctgtgga acgtcgtgcc atgc tgtagc gaagaaagca tggttgctgc ggcaggtcag ccaatgtagc gtcccaccct aggatgtgcc ttcagacaag ggggaggaac cttatgggtg tccctggggt ggctctgaga tggt tcacgc tctggagttc aaattagcca gagaatcgc t gcatgccagc gggcatggtg aggttgaagt agaccctgtc tgctctacac agtgataatg taaaaggccg cccagcccac gttaggtgc ctgacctttg gcccaggacc gcctgggaag ctgtgtccgc cccaggctct gcccacagtg tctgcctttt ttcatgcctg agttcgagac aggcgtggtg gcttgaacct gggcgacaga gggcagtgaa aaggcctcat gggatggcgc ttccagacac ggggatgttg atgtgctctg agcacgtgac cgtgcaccag gcagttccca aagagctttt taaaatgggg cccaggctgg cgccattctc cggctaattt C tcgatct CC catgagccae gactgggcaa tggcatgcac aggaggtcaa gaccctggct gcatcagggc acaagtgaaa tggacaggac atagtgccaa ggccctcag 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180 12240 12300 12360 12420 12480 12540 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 13260 13320 13380 13440 13500 13560 13620 13680 13740 13800 13 860 13920 13980 14040 14100 14160 14220 14280 14340 WO 01/21647 WO 0121647PCT/IBOO/01470 acacaacact tgagatgcgt aggactgcat aaaaaaaatg tgttcagatt agccctagct agctgcagtg tatttcgtgg ggagcccttt tagagccctc ttatgcaatg ccatgcrcta ccagcctgga ctgtttctct tcaggcgggg ccatacatca cactgtggac gctgggccta tggcacctgc ggggc tgcag acctggggga ccc agcct ct tcgtccccga tcctcccag tgtggtatgc ctgcccgcac cccgaggc aagggaagga aatttagcca ttcctccaac atacacatgg taaaaaaacc ggccaagacg aaacccagtt tcccagctac agtgagc tga aaaaaaaaag actacaigat tccaggcagt cctcccgcct attttttgat aaactcctgg catgtgccac ttgcccaggc atgggattac ttttggagac ctgcaacctc gatataggcg tgccatgttg cctaccttgg ttaagcaaat tgtctagggt agatgagagc acagaciagag tgctcctgtc agctgattga agtggcactg gaccacagct tgtgcacaca caggtggaga ctccctagg tgggtgcatc atataaaata ggtggggctc gaatgtgaca atgactttca tgtgattatg tgtaaagtaa acttacccca agccatgatc gccctaggtc ggggtgctaa agaaccccct ggagtttctt gggcttcagc gctgggcttt tttgtccctc cccatagaag cctactgctt ccctcactaa ggctaggctg ggtgctcttg tctgtctccc gggccgtaac ggcacaaaaa cctcagtgct gcctccgggc ctggctgcct acttgctgct cgtaagtgtc ggtggccatc gtggggagaa ttttagttta taaaaatgtc aaatcttggc agtggatcac ctctactaaa tcaggagact gatcacgcca aaaaaaaaat ttaagcaaaa gcagtgcgat cagcctctgg ttttgttttt cctcaggtga catgctggcc tggtcttgaa aggcatgaac agtttcactc cgccccccgg cctgccacca gccaggctgg cctcccaaag gtttgaaaac cgtatc tagc t aaaggggaa agggacctaa tctcttctgt cgtggtcaca atggacactt gttgccagtc gtccaagata gctgaggcct gctcagtgtc tggggatttg ttctacgtct agagctccag gtgcatgagt tatgtgggta ttgtcacaaa aattaaacca gaggctgagg acacccctgc cctgtgcctg gacctatgaa gggatttgta acctataagc c ccc agcc cc cgggggtggc cagggaggac gtacgggggg ctgactctag cctggcctga ggctgaggag agggtgtggc tctgtgcagg tggggaatgg atccagccag ggct ccgca c tcagggcctg tcctcatctt gctacgtcag ccgc tcatgg cacctgcccc agtaggctga tttttattta caacagtaca taggcgcagt t tgagg tcag aatacaaaaa gaggcatgag ctgcactcca gttaagtgaa aaaatttttt catagctctc agcagctggg tgtagagacg tcctccgaag aatttttaaa ctcttgacct taccacacct tgtcgcccag gtttaagcta cgcctgacta tctcgaactc tgttaggact acatacccac acaccagcat gtaagcaaac ctctatgaga gaatgtttgt gccatcagcc cagcatccct cttggtttcc catgaacagt gcttgacacc atcccctata gcgagctcat gtcagctgct gggcactcga agagatggag tttttgcctt gagtttattc tttcagccag tgggaggatc actccagact ctggaacagg tgagggaaac ttggagccct acccacatgt ttcccgcttc acagcctggg ctcaggggtg tggatcctga ttagtatccc ctgtggctct agcctggggt gtctgggcag ctgcctcgtt ccgggatggt ggctgcaggt catgtacccc ttgtctttct cctcctcctg gtgcccctgc ccaccctggt caggacagtg ggagggtctg tgttgttctt agatactagt gattacgcct gagttccaga ttagctgggc aatcgcttaa gce tgggcga aaagttaaga ttgttttaga tgcagcctca actgtaggca gggtctcagt tcagcctccc aattttctgt caagtgatcc ggccttaaac.

gctggagtaa ttctcctgcc atttttgtat ctgacctcag acaggtgtga aggaatgctg ggctactgtg caagcagggg egagtcagac gaatatccca ttgggacacc ctagctgctg ttcaggaggg gaagtgccag ttcctgctca aaatggggct gtgcacacca gggcactgcc gggaagacaa accttgtggg c atgggt c CC ctgtatattg gtgtggtgac gcctgagccc gggcgacaga acatccctat ttagggtgcc cgtggcataa gggcgggtgg agggcacacc ctggctctgg gc tgagctct gt tgggct tc cttccccact gaggcatcta gcaggccagg ctggctctct ttctgccttg agaatgggga tccttggtga tgctgtgccg gcagactggc ggcatytgct tgcccagaca ttgggcaaca gcgttggtct ctgtttagat ttcttttgta cacatggaag gtaatcccag tcagcctggc atggtggtga acccaagaag cagagtgaga aaccaaacaa gaaagggtct aactcccggg cacaccacca atgttgccca caaagtgctg agagacaggg tgcctcaggc ttaagcaaat agtggcgtga tcagcctccc ttttagtaga gcaatccgct gccaccatgc cacattttac gagage tggg aaggtaagag atgtgcaatt acgggacact agaccacagt tcccgtttcc tgtctgggta gcaatccttg gaagcccagt tatggcagag ttgagcatgg actatcagcc gaaccggctc tcccagaacc ttcctgtttt tgttaatttg acatgcctgt acgaggt tga gctgagatcc caccgtggtt caagctgagg cacaggtgga agggtaggag ttgcacttgg cc agcat aa t tacctggttt tcrggagctc aaaccctgct gtggtctggc gctctgtgac ctttggtctg tgttttttgc gtgtgctgtg gctttgcaaa ttagccctgt tcttcgtggt ggtgccagtg agtgctgctg tcctgcatcc ggagggtgtg tgtcgtttac agtataatcc taaagccctc cactttggga caacatggta tcgcctgtaa tggaggttgc ctctgtctca ggtttacaac cattctgtca ttcaagcagt tgcccagcta gcctgat etc ggattacagg tcttgctatg tcccaaagtg tttttttttt tctctgctca gagtagctgg gacggggttt cccccgcacc ctggccaaa t ccagctacta actggatgtg aagacagaag gaaaaagact tt cacagagg gtgtacacta cctcctcggg gaccagcctg caagcatggg gagcagtttc ctcaccacac ggcc caacc t tcagtagtga 14400 14460 14520 14580 14640 14700 14760 14820 14080 14940 15000 15060 15120 15180 15240 15300 15360 15420 15480 15540 15600 15660 15720 15780 15840 15900 15960 16020 16080 16140 16200 16260 16320 16380 16440 16500 16560 16620 16680 16740 16800 16860 16920 16980 17040 17100 17160 17220 17280 17340 17400 17460 17520 17580 17640 17700 17760 17820 17880 17940 18000 18060 18120 WO 01/21647 WO 0121647PCTIIBOO/01470 ctgagggaca cttccctctc attaaggaag Ctcaaaggat ccatcccctc gtccatttca ggccaggact catccggaga tacatctgct atgataaagt tcttctgctt cattttcctg gcctcttgcc agcctggtg ctttgctgct tctggaaggt acaggggctt cagcatctgg tggccaCttc gacaaatgtt gtgggtgcct agctggtgtc agatgtccaa tccctCCgga ttgattrtgt tttatgcccc tgattcccat ggartagctc tggacactga CtCCtggtgc acccctgctt ttggatgtgc ccccCCtgtc Ccatgcgggt gcccccccag aggtgggggg tggaccccgg tcacctccct cgatccggga agcccgccag tggacgccct cgagtaatgg acctctatga acttCaggtC accCtcggga aggctgtgag aggcctacta agcgcaggct cagccggtcc tcgggaaagt aatttgaagg atCaCaagct cccgtatgcg gagacccagc ccaccgcccg tcCgccttcg agccatccc CCtggccccc gtttcaccag ccgcccgccc t ccgccccgg gaggaaaaaa cggactgggc gggcaccagt tgagtcCtgt tcgctggcta cacttCCCtg cctcttgttg cagatgggct aaatgctggt ccctgttac gctgccacag tgcaggctgC ccaaaccaaa ttggggtccc ctagggagga ggcaggcaga tggccggggc gaccaggtgg ggctgccaaa gctttatata ttccagtaag ggcgaattac gcgtgcaccc cccc agcct C ggaagctcta acccaatggg Cctcacagtg CagCaCCtat gggCCCtgCC aggtgaggcc ggggcagggg gcggccactg cgggacacgg Ctgtaaggga cctagaagtc cctgtactac tggCcgtgtg gtgaaacatg gctcctccag ccacgaggac tgaggagtgg ggagcaggca ggacgacctC tgggaggaga ccaagacgac tcgggagcgc caacggctcc gaagaagggg cccgcccgcg caagaaggtg ccgcggct ca ttagtcgtct aacactgatg ctggagagaa tCtgtaaagc ctagcccagc CCtgcgtgcg cgcaccgcag Ccccagcccc gacgacccca ggcggcgcgc ccggagacag gctcgcgccg aaaaggggga caagccggge cagagCcctg ttccttctgt attttatagt aaaaaattcc tgttcagctg gcaaccaagt cccaatgcca ccaacccagg cctccgccca ctgagctaat tcagaggtga agggaggaag aggaagggag aatgggtgca aggcaagt ta ggcaggaccc gaggatactt aagggcagct ttagctgggg catgctgtta tctgaaaaat agggatgctg cagcggagag tatggggcag tatgccgccg gcccacctgt tacaacgggt gggggaagca ctggaaggaa acagccactc acagcagtgt gaggggtggg cgcagtggct atggagaagg gagcggggta tctccctgat cagtcagtga gactggcgat ggtggccact ggcgggggct accccgccga agccgggcct tcgagggact CC tcc tgc cg aggtccgggg tcggaggaga ccgcccccgt agggccgccc tacccttctt gatctgacgt aagccctgcc ccatttgttc gCC9Cgtccc ccgcoCCCgc CgCCCCggCC gctggccgcc caccgacct gCCCCtgCtc ggcgacctcg acagcgcgcg ccgccgccgc aaaatagaaa ggcggccgcg gtgcacacag taggtcctga cattgaagtc actgctccct acagcttttt ttgcagtgaa Cattccctg gcccattaa cccttcagga gaaggggctt ggca CCCt Ct ccaCttgcct gaggatctta gtccatgagt attcccgagg acgtctccca gatt tCggCt ttgttgccct agttggaagt aatgaaacat cacacaggaa Cagaaatgga gacggcttgg CCtggCtcct gcaaagcagc ctccgccaa accctggagg ggaacagctg gagtgtcttg t CCC CCagc t ggcCtCtggt ccaggatcca acaggattca agctggccaa agcaggagcc acctgccgca cacccccctt ctcggCCttc ccccccggag ggcgggccag gcaccgccga acatgccccc tCCCaCgctC ac C Ccagg tC acctccccta ggaggagacc actcggagac tccctggcgt tCtttCtccc tttctacgta atacccctcc ggccgcgcgg gggggcaccg gC CgCCggcc ccgcccaggc gggagcgcgc actttactgt cgggtacccc CgCCCCgCgg cgctcccggg cgccgccgcg gcggcggcgg cgagccggcg agtgacccca cttcatgggt agtggtgtgc ggaggcttat gctcagtgag cccac taaga tccccacacc ctccctggca ggcagcaggt cctctaggct ctgggcccat agggcccagg ccttgaactg tagaaacact ctcctgccac gatgactcat tgtggggaca gtaaacacac ttaggtaaaa tgttctgcca gtggggtggg atgaggacca gaaggaggtc gcctcatccc CaCC tcagg t gaccccaccc at acc t gga gtgggmgtgt ggagccgagg ggtggccaag gagaatccat tcetcccaaa ggccagccag cttcgaccct ttggggtctg gggactcttg ccctgcagcc CCggggCCCt tcccagggga gcggccccgg gtcagggagc gcggagccgc ccgggaC CCc ccaccaccac tgatgggcgg ccacaaggag cgactcgcag ccagaccgtc ttgcagaact gcttttgkat cgagtctaat ggcgggggac gagtccgggg ggaqacgtg gtgcgcatgc gcgcgctcct ctccaaactc gacgttccat agccccgggc ccgcctcccc cgcgcgcagc ctgcagcagc atccagggca gagaagcagc tgttgttagc aacctggttc gcaggccatc taagtgttag ccagagctag acatttcctc gaggccctgt cccaCtgctg gtgcacttag cLtctcctC aattttgcaa tcaagcctag agaggagaca tgcatCtcaa tttttctaga gtggtggac agaccatggg ccttttgatt CCCtggggct gtctctgtga acagggactc caggcccagg ccttctcctg gttcccagca ccaccagcta gacgttgaca gctgggcatc aggggctctg gctcctatgt cgtcccgaag ccgacCacca caggacgact tctcgacstg agggctttta gtgcaaaccc atgagtgaag gccctcaccc tgggaccagg gCCCgctccg aggt ctCC Ca agccgggacg cactacgacg cgtacccggg ctactggagg gaggaggaag gcgtCCcgag cctgggcccc tggCCC tgag tttttttttt aaaacgtata cggggctgct ccgggaggaa ccccgcgCag gCCCCggCCC ctccccttCC gggcagccca ccagacccgc tcgcgcgcgc ccagcgcgcg tcaagtaaag gatccgccgc ctggCggCgg 18180 18240 18300 18360 18420 18480 18540 18600 18660 18720 18780 18840 18900 18960 19020 19080 19140 19200 19260 19320 19380 19440 19500 19560 19620 19680 19740 19800 19860 19920 19980 20040 20100 20160 20220 20280 20340 20400 20460 20520 20580 20640 20700 20760 20820 20880 20940 21000 21060 21120 21180 21240 21300 21360 21420 21480 21540 21600 21660 21720 21780 21840 21900 WO 01/21647 ccagccaggg ggaggcctga gcgtcccgga gctgcagcgg atgaccccac tcctgccgcg tccntccntc c ccc atgga c cgccaggtaa cggcctgcag gccgccggcg cccggcctca cgcgggcgcg acccgaggcg cccggggagg caggcgggga aggcggcgtt aggtgagcgg gacctggccc gtccaggtga gtgtccaccg gcagcccagc PCT/IBOO/01470 cgggccgtgt gggccgcgtg gcctcgggcc ctgcaggagc tgtttgttccc cggcgtgagc ccctcccctc atgctggacc gatccccggc gccggggccg cgggggggac ggcgcggccg ggccgcgctg gaggagggcg gc tgggggcg cggcccagga cggcgaccac cgggccgcga cagcgccggc ctgatggtca ctgccttcgc gcccgggccc tcaaaaaaaa cagcgggcgg gcccggagcc ggcggcggct cgtgatcagc gccgggnc tc ccctcccccc cgggtctgga ccggccgtgc ccatgatccc ctggcgcctc ggtgggactg accctgctcc tcgagctgca ctcggcgcgg gcggaggagc aacatccagt gagcgaacgg ctcgccgctc gctggacggc gggggggcag cgccgct aagtcgcggc gcagctgggt ggcgggcggg gcngcggcgg gcgagcggct ggggcccccc cgggccccgc tcccgctgcc ccccgcgccc gagcggccgc ccgcccccgg gggccctgca ctcctgtgcc ggaaggtgag ccctgaccgt agacagcggt accagttccg gcgggcgggc tgccgccccc cagggcgaca caggctgtga ggcggcggct gggc tggggg cggaggcgga cngcggcatc cccgtatctc cggccgCCCg gccccccCCg tcggccaccg cggccccggc gggccccgct cccccggcct gc tgggcgcg cctggcagcc tgcttgccgg gccccgaccc ggccatcacc cacagagaca gggcgcgccg tgcaggtgac cagc tggcgc cccaggtggg gctcagggaa cggccgcgcg ggcggcggcg tcctcctcac ctccgtcccc ccccctcccc cccccgcccc ctgctgccgc cccggccccg caaaatggag cggcggcgcc g9g9c99g9g acgacaaggg gccggccgcg tcctcggccc agcgtccagc aatggaggac ggaaggctcg ataccgcgta cgtcagcgtc tgtggacggg 21960 22020 22080 22140 22200 22260 22320 22380 22440 22500 22560 22620 22680 22740 22800 22860 22920 22980 23040 23100 23160 23187 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 2 2158

DN~A

Homo sapiens allele 595 9-3 -324 allele 940 9-6- 187 allele 1191 9-7- 325 allele 1362 9- 9-246 polymorphic polymorphic polymorphic polymorphic base C or T base C or T base A or G base G or C allele 1658 LSRX9f 13 BM allele 2079 LSRX9f14 -EM polymorphic base deletion of AGO polymorphic base T or G <400> 2 tggagtgtgg ctcggaggac cgcggcgggt caagcacctt tctcccccat atctgaaagc atgccctttg tccacgtcgt ttacgCtcat taaaacttcc aga atg caa cag gac Met Gln Gin Asp WO 01/21647 WO 0121647PCT/IBOO/01470 ctt gga gta ggg Leu Gly Val Gly agg aac gga agt ggg aag ggg agg agc Arg Asn Gly Ser Gly Lys Gly Arg Ser ccc tcC tgg Pro Ser Trp tgg tgc gcg ccg Trp, Cys Ala Pro ccc cta agg tac Pro Leu Arg Tyr ttt gga Phe Gly agg gac gcg Arg Asp Ala gcc ggc ggg Ala Giy Gly cgg gac gcg Arg Asp Ala aga cgc gcc cag acg gcc gcg atg Arg Arg Ala Gin Thr Ala Ala Met ctc tcc aga ggg Leu Ser Arg Gly tcc cac ccg Ser His Pro gcg ctg ttg Ala Leu Leu gcc gca ggc Ala Ala Gly tgg tgc aca Trp Cys Thr gtc gtc ttc Val Val Phe ctt ctg ctt Leu Leu Leu gct cet Ala Pro gcc agg gcc Ala Arg Ala gtg acc gtg Val Thr Val ccc tac cac Pro Tyr His atc ctc ttc Ile Leu Phe gtg acc ctg ccc Val Thr Leu Pro acc tac cag Thr Tyr Gin atg acc Met Thr 115 tcg acc ccc Ser Thr Pro cgg gac cgc Arg Asp Arg 135 caa ccc atc gtc Gin Pro Ile Val aag tac aag Lys Tyr Lys tct ttc tgc Ser Phe Cys 130 gac aac cag Asp Asn Gin gcc gat gcc Ala Asp Ala ccg gcc agc Pro Ala Ser ctc aat Leu Asn 150 gag tgc Giu Cys gcc cag ctg gca gcc ggg aac eca ggc Ala Gin Leu Ala Ala Gly Asn Pro Giy aac ccc tac gty Asn Pro Tyr Val cag gac agc Gin Asp Ser acc gtc agg Thr Val Axg gcc acc aag Ala Thr Lys 163 211 259 307 355 403 451 499 547 595 643 691 739 787 835 883 931 979 1027 1075 1123 aac gct gtg acc Asn Ala Val Thr gga gat tac Gly Asp Tyr cag ggc cgg agg Gin Gly Arg Arg att acc Ile Thr 195 ggg gac Gly Asp atc acc gga Ile Thr Gly agt ggt gtg Ser Gly Val 215 aac aat gag Asn Aen Glu gct gac ctg acc Ala Asp Leu Thr cag acg gcg Gin Thr Ala tat tac tgc tcc Tyr Tyr Cys Ser gtc tca gcc cag Val Ser Ala Gin ctc cag ggg Leu Gin Gly acc tca ggg Thr Ser Gly 9CC tac gca Ala Tyr Ala 230 gtg gct Val Ala gag Glu 235 ggt Gly atc gtc ctt Ile Val Leu gag ctc tta Giu Leu Leu ttt cag gcg Phe Gin Ala ata gaa gac Ile Giu Asp 245 ctc Leu ttc gtg gtt Phe Val Val tgC ctg gct Cys Leu Ala ctc atc ttc Leu Ile Phe ctc ctc Leu Leu 275 tgc tac Cys Tyr ctg ggc aty Leu Gly Ile gtc agg tgc Val Arg Cys 295 tat gcc gcc Tyr Ala Ala tgc cag tgc Cys Gin Cys cac act tgc His Thr Cys tgc tgc cca Cys Cys Pro tgc tgc tgc Cys Cys Cys gag gcc ctg Glu Ala Leu att tat gcc Ile Tyr Ala ggc aaa gca Gly Lys Ala acc tca ggt gtt Thr Ser Gly Val tct ccc gcc aag Ser Pro Ala Lys 335 acc tat gcc Thr Tyr Ala cca ccc cca Pro Pro Pro WO 01/21647 WO 0121647PCTIiBOO/01470 get atg att ccc atg ggc cct 9CC tac Ala Met Ile Pro Met Gly Pro Ala Tyr ggg tac cet gga dly Tyr Pro Gly gga tac dly Tyr 355 tee tat Ser Tyr ect gga gac Pro Gly Asp gta ccc ctg Val Pro Leu 375 gac agg art age Asp Arg Xaa Ser get ggt ggc caa Ala Gly Gly Gin egg gac aeg Arg Asp Thr age gtg gec tct gaa gte cgc Ser Val Ala Ser Glu Val Arg 385 age. ggc Ser Gly 390 tac agg att cag Tyr Arg Ile Gin agc cag cag gac Ser Gin Gin Asp tcc atg egg gte Ser Met Arg Val tac tae atg gag Tyr Tyr Met Giu gag etg 9CC aac ttc gac cct tet cga Glu Leu Ala Asn Phe Asp Pro Ser Arg 415 gge ccc ccc agt Gly Pro Pro Ser etc cac gag gac Leu His Glu Asp 440 ace ecg ate egg Thr Pro Ile Arg 455 agg gga egg gac Arg Gly Trp Asp 470 egg gee agg egg Arg Ala Arg Arg 485 ace ceg ccg age Thr Pro Pro Ser cgt gtg gag egg Arg Val Glu Arg atg age gaa gte Met Ser Glu Val ace tee Thr Ser 435 tgg ega tct.

Trp Arg Ser tcc egg ggc Ser Arg Gly gat gag gag Asp Glu Glu ggc cac tee Gly His Ser ecc gec etc Pro Ala Leu 450 egg agt c Arg Ser Pro 999 ggc tg Gly Gly Trp eag gag ccc gee agg gag cag gca Gin Glu Pro Ala Arg Glu Gin Ala ec Pro gee ege tee gtg gae gee etg gac gac Ala Arg Ser Val Asp Ala Leu Asp Asp gee gag tea ggg Ala Glu Ser Gly tet ccc aeg Ser Pro Thr agt aat Ser Asn 515 1171 1219 1267 1315 1363 1411 1459 1507 1555 1603 1651 1699 1747 1795 1843 1891 1939 1987 2035 2083 2143 2158 ggt 999 aga Gly Gly Arg gac etc tat Asp Leu Tyr 535 ccc eac tac Pro His Tyr gee tae atg Ala Tyr Met egg age egc Arg Ser Arg eaa gac gac Gin Asp Asp gac ttc eca Asp Phe Pro age egg gac Ser Arg Asp 530 tee egg gac Ser Arg Asp gee gae ccc Ala Asp Pro gac gae tte Asp Asp Phe eac cac egt His His Arg 570 agg Arg 555 ace Thr egg gag ege Arg Glu Arg agg Arg 565 cac His egg gac ect Arg Asp Pro gac aac gge tee Asp Asn Gly Ser tee ggg gac etc Ser Gly Asp Leu eec tat gat ggg egg eta ctg gag gag get gtg agg Pro Tyr Asp Gly Arg Leu Leu Glu Giu Ala Val Arg 585 590 595 aag aag 999 Lys Lys Gly gag gee tac Glu Ala Tyr 615 gag gag agg agg Glu Glu Arg Arg ccc eac aag gag Pro His Lys Glu gag gag gaa Giu Glu Giu 610 ace gac teg Thr Asp Ser tac ceg ccc geg Tyr Pro Pro Ala ccc eeg tac teg Pro Pro Tyr Ser eag geg tee ega gag ege agg etc aag aag aac ttg Gin Ala Ser Arg Glu Arg Arg Leu Lys Lys Asn Leu 630 635 640 9cc etg agt egg Ala Leu Ser Arg gaa agt Glu Ser 645 tta gte gte tga Leu Val Val 650 tetgacgttt tctacgtagc ttttgkattt ttttttttaa tttgaaggaa cactgatgaa geeetgecat aeccetceg agtctaataa aacgtataat cacaa WO 01/21647 WO 0121647PCT/IBOO/01470 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> 3 649

PRT

Homo sapiens

VARIANT

363 9-7-325 polymorphic

VARIANT

420 9-9-246 polymorphic amino acid Ser or Asn amino acid Pro or Arg <220> <221> V <222> 51 <223> U~ <400> 3 Met Gin 1 Gly Arg Arg Tyr Met Ala Ala Ala Thr Trp Pro Tyr Tyr Gln Lys Ser 130 Val Asp 145 Asn Pro Ala Thr Arg Arg Ala Trp, 210 Asp Leu 225 Arg Thr lie Glu Ile Phe Cys Cys 290 Pro Glu 305 Ser Ile kRIANT L9 ;RX9f 13 -BM polymorphic amino acid deletion of Arg Asp Val1 Gly Leu Gly Thr Val 100 Thr Cys Gin Val Gin 180 Thr Asp Gly Gly Tr_- 260 Leu Tyr Leu Al a Thr Trp Arg Arg Phe Ile Pro Pro Asp Ala Val 170 Leu Asp Cys Ala Pro 250 va 1 Cys Cys Ala His 330 Ser Arg Thr Ser Leu Val Pro 110 Trp Pro Pro Arg Tyr 190 Asp Ser Val Ala Ala 270 Pro Cys Gly Lys Leu Ala Pro Ser Asn Thr Tyr Ser Tyr 160 Val Gly Thr Gln Gly 240 Pro Leu Thr Cys Pro 320 Thr Pro Ala Lys 335 WO 01/21647 WO 0121647PCTIBOO/01470 Pro Pro 340 Gly Tyr 355 Ser Tyr Val. Arg Arg Val Arg Pro 420 Thr Ser 435 Ala Leu Ser Pro Gly Ti-p Asp Leu 500 Ser Asn 515 Arg Asp Arg Asp Asp Pro Ser Arg 580 Val Arg 595 Glu Glu Asp Ser Ser Arg Ala Met Ile Pro Pro Gly Asp Val 360 Val Pro Leu Leu 375 Ser Gly Tyr Arg 390 Leu Tyr Tyr Met 405 Gly Pro Pro Ser Leu His Glu Asp 440 Thi- Pro Ile Arg 455 Arg Gly Ti-p Asp 470 Arg Ala Arg Arg 485 Thi- Pro Pro Ser Gly Gly Arg Ser 520 Asp Leu Tyr Asp 535 Pro His Tyr Asp 550 Arg Ser His His 565 Ser Gly Asp Leu Lys Lys Gly Ser 600 Giu Ala Tyr Tyr 615 Gin Ala Ser Arg 630 Giu Ser Leu Val 645 Met 345 Asp Arg Ile Glu Gly 425 Asp Asp Gin Pro Thr 505 Arg Gin Asp His Pro 585 Glu Pro Glu Val1 Gly Tyr Giy Gly Val Ala Asp Asp 400 Phe Asp 415 Met Ser Ser Arg His Ser Gin Ala 480 Asp Ala 495 Arg Ser Arg Ser Phe Pro Arg Pro 560 Arg Asp 575 Leu Glu His Lys Tyr Ser Asn Leu 640 <210> 4 <211> 2101 <212> DNA <213> Homo sa <220> <221> allele <222> 595 <223> 9-3-324 <220> <221> allele <222> 8e3 <223> 9-6-187 <220> <221> allele <222> 1134 <223> 9-7-325 <220> <221> allele piens polymorphic polymorphic polymorphic base C or T base C or T base A or G WO 01/21647 29 <222> 1305 <223> 9-9-246 :polymorphic base G or C PCTIBOO/01470 <220> <221> <222> <223> <220> <221> <222> <223> allele 1601 LSRX9f13-BM allele 2022 LSRX9fl4-BM :polymorphic base deletion of AGO :polymorphic base T or G <400> 4 tggagtgtgg ctcggaggac cgcggcgggt caagcacctt atgccctttg tccacgtcgt ttacgctcat taaaacttcc gga. ctt gga gta ggg aca. agg aac gga aqt ggg Gly Leu Gly Val Gly Thr Arg Asn Gly Ser Gly tctcccccat atctgaaagc aga atg caa cag gac Met Gin Gin Asp aag ggg agg agc Lys Gly Arg Ser cac ccc tcc tgg His Pro Ser Trp tgg tgc gcg ccg Trp Cys Ala Pro ccc cta. agg tac Pro Leu Arg Tyr ttt gga Phe Gly ctg ttg Leu Leu agg gac gcg cgg gcc aga cgc gcc Arg Asp Ala Arg Ala Arg Arg Ala acg gcc gcg atg gcg Thr Ala Ala Met Ala gcc ggc ggg Ala Gly Gly cgg gac gcg Arg Asp Ala ctc tcc aga. ggg Leu Ser Arg Gly ggc tcc cac ccg Gly Ser His Pro gcc gca. ggc Ala Ala Gly tgg tgc aca Trp, Cys Thr gtc gtc ttc Val Val Phe tgg ctt ctg ctt Trp Leu Leu Leu gct cct Ala Pro agc Ser aac Asn gcc agg gcc Ala Arg Ala gtg acc gtg Val Thr Val ccc tac cac Pro Tyr His atc ctc ttc Ile Leu Phe gtg acc ctg Val Thr Leu acc tac cag Thr Tyr Gin atg acc Met Thr 115 115 163 211 259 307 355 403 451 499 547 595 643 691 739 787 835 tcg acc ccc Ser Thr Pro cgg gac cgc Arg Asp Arg 135 ccc atc gtc Pro Ile Val aag tac aag Lys Tyr Lys tet ttc tgc Ser Phe Cys 130 gac aac cag Asp Asn Gin gcc gat gcc ttc Ala Asp Ala Phe ccg gee agc gtc Pro Ala Ser Val ctc aat Leu Asn 150 gee cag ctg gca gcc ggg aac cca ggc tac aac ccc tac gty Ala Gin Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val 155 160 tgc cag gac agc Cys Gin Asp Ser cgc ace gte agg Arg Thr Val Arg gtg gee aec aag Val Ala Thr Lys aae gct gtg Asn Ala Val etg gga. gat tac Leu Gly Asp Tyr cag gge cgg agg Gin Gly Arg Arg att ac Ile Thr 195 atc acc gga. aat Ile Thr Gly Asn 200 get gac ctg ace Ala Asp Leu Thr gac eag acg gcg Asp Gin Thr Aia agt ggt gtg tat tac tyc tce Ser Gly Val Tyr Tyr Cys Ser 215 aac aat gag gcc tac gca gag Aen Asn Giu Ala Tyr Ala Giu 230 235 gtc tca gcc cag Val Ser Ala Gin tgg ggg gac Trp Gly Asp 210 cte cag ggg Leu Gin Gly cte ttc gtg Leu Phe Val ctc ate gtc ctt Leu Ile Val Leu WO 01/21647 WO 0121647PCT/IBOO/01470 gtg gta tgc ctg Val Val Cys Leu gcc ttc ctc atc Ala Phe Leu Ile ctc ctc ctg ggc Leu Leu Leu Gly tgg tgc cag Trp, Cys Gin tgc ccg cac act Cys Pro His Thr tgc tgc tac gtc Cys Cys Tyr Val ccc tgc tgC Pro Cys Cys ggc aaa gca Giy Lys Ala 295 tat gcc cac Tyr Ala His aag tgc tgc Lys Cys Cys ccc gag 9CC ctg Pro Glu Ala Leu tat 9CC gcc Tyr Ala Ala 290 ccc agc acc Pro Ser Thr gcc acc tca. ggt gtt Ala Thr Ser Giy Val agc att tat Ser Ile Tyr ctg tct ccc Leu Ser Pro aag acc cca ccc Lys Thr Pro Pro cca gct atg att Pro Ala Met Ile 310 ccc atg Pro Met ggc cct gcc Gly Pro Ala ggg tac cct Gly Tyr Pro gga tac cct gga Gly Tyr Pro Gly gac agg art Asp Arg Xaa tca gct ggt ggc Ser Ala Gly Gly ggc tcc tat gta Gly Ser Tyr Val ccc ctg Pro Leu 355 ctt cgg gac Leu Arg Asp agg att cag Arg Ile Gin 375 atg gag aag Met Giu Lys 390 gac agc agt gtg Asp Ser Ser Val tct gaa gtc cgc Ser Glu Val Arg gcc agc cag cag Ala Scr Gin Gin tcc atg cgg Ser Met Arg agt ggC tac Ser Gly Tyr 370 ctg tac tac Leu Tyr Tyr ggc ccc ccc Gly Pro Pro gag ctg gcc Giu Leu Ala gac cct tct Asp Pro Ser cga Arg 400 ggc cgt gtg gag Giy Arg Vai Giu gcc atg agt gaa Ala Met Ser Giu acc tcc ctc cac Thr Ser Leu His 883 931 979 1027 1075 1123 1171 1219 1267 1315 1363 1411 1459 1507 1555 1603 1651 1699 1747 1795 1843 gac tgg cga Asp Trp Arg tct Ser 425 cgg cct tcc cgg ggc cct gcc ctc acc Arg Pro Ser Arg Gly Pro Ala Leu Thr 430 ccg atc Pro Ile 435 cgg gat gag Arg Asp Giu gac cag gag Asp Gin Giu 455 cgg ccc cgg Arg Pro Arg tgg ggt ggc cac Trp Gly Gly His ccc egg agt ccc Pro Arg Ser Pro gCC agg gag Ala Arg Glu gca ggc ggg ggc Ala Gly Gly Gly agg gga. tgg Arg Gly Trp 450 cgg 9CC agg Arg Ala Arg acc ccg ccg Thr Pro Pro gcc cgc tcc Ala Arg Ser gcc ctg gac Ala Leu Asp gac Asp 480 470 agc acc Ser Thr gcc gag tca Ala Glu Scr agc agg tct ccc Scr Arg Scr Pro agt aat ggt ggg Ser Asn Gly Gly cgg gcc tac Arg Ala Tyr ccc ccg cgg agc Pro Pro Arg Ser agc cgg gac gac Ser Arg Asp Asp ctc tat Leu Tyr 515 gac caa gac Asp Gin Asp gac gac ttc Asp Asp Phe 535 cac cac cgt His His Arg 550 agg gac ttc Arg Asp Phe cgc tcc cgg gac Arg Ser Arg Asp ccc cac tac Pro His Tyr 530 agg tcc cac Arg Ser His tct cgg gag Ser Arg Giu cct cct gcc gac Pro Pro Ala Asp acc cgg gac Thr Arg, Asp cgg gac aac ggc tcc agg tcc ggg gac Arg Asp Asn Gly Scr Arg Ser Gly Asp ccc tat gat ggg Pro Tyr Asp Gly eta ctg gag gag Leu Leu Glu Giu gtg agg aag aag Val Arg Lys Lys WO 01/21647 PCT/IBOO/0 1470 31 tcg gag gag agg agg aga ccc cac aag gag gag gag gaa gag gcc tac 1891 Ser Glu Glu Arg Arg Arg Pro His Lys Glu Glu Glu Glu Giu Ala Tyr 585 590 595 tac ccg ccc gcg CCg ccc ccg tac tcg gag acc gac tcg cag gcg tcc 1939 Tyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu Thr Asp Ser Gin Ala Ser 500 605 610 cga gag cgc agg ctc aag aag aac ttg gcc ctg agt cgg gaa agt tta 1987 Arg Glu Arg Arg Leu Lys Lye Asn Leu Ala Leu Ser Arg Glu Ser Leu 615 620 625 gtc gtc tga tctgacgttt tctacgtagc ttttgkattt ttttttttaa 2036 Val Val 630 tttgaaggaa cactgatgaa gccctgccat acccctcccg agtctaataa aacgtataat 2096 cacaa 2101 <210> <211> 630 <212> PRT <213> Homo sapiens <220> <221> VARIANT <222> 344 <223> 9-7-325 polymorphic amino acid Ser or Asn <220> <221> VARIANT <222> 401 <223> 9-9-246 polymorphic amino acid Pro or Arg <220> <221> VARIANT <222> 500 <223> LSRX9f 13-EM :polymorphic amino acid deletion of Arg <400> Met Gin Gin Asp Gly Leu Gly Val Gly Thr Arg Asn Gly Ser Gly Lys 1 5 10 Gly Arg Ser Val His Pro Ser Trp Pro Trp Cys Ala Pro Arg Pro Leu 25 Arg Tyr Phe Gly Arg Asp Ala Arg Ala Arg Arg Ala Gin Thr Ala Ala 40 Met Ala Leu Leu Ala Gly Gly Leu Ser Arg Gly Leu Gly Ser His Pro 55 Ala Ala Ala Gly Arg Asp Ala Val Val Phe Val Trp Leu Leu Leu Ser 70 75 Thr Trp Cye Thr Ala Pro Ala Arg Ala Ile Gin Val Thr Val Ser Asn 90 Pro Tyr His Val Val Ile Leu Phe Gin Pro Val Thr Leu Pro Cys Thr 100 105 110 Tyr Gin Met Thr Ser Thr Pro Thr Gin Pro Ile Val Ile Trp Lys Tyr 115 120 125 Lys Ser Phe Cys Arg Asp Arg Ile Ala Asp Ala Phe Ser Pro Ala Ser 130 135 140 Val Asp Asn Gin Leu Asn Ala Gin Leu Ala Ala Gly Asn Pro Gly Tyr 145 150 155 160 Asn Pro Tyr Val Glu Cys Gin Asp Ser Val Arg Thr Val Arg Val Val 165 170 175 Ala Thr Lys Gin Gly Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gin Gly 180 185 190 Arg Arg Ile Thr Ile Thr Gly Asn Ala Asp Leu Thr Phe Asp Gin Thr 195 200 205 Ala Trp Gly Asp Ser Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gin WO 01/21647 PCTTBOO/01470 32 210 215 220 Asp Leu Gin Giy Asn Asn Giu Ala Tyr Ala Giu Leu Ile Vai Leu Asp 225 230 235 240 Trp Leu Phe Val Vai Val Val Cys Leu Ala Ala Phe Leu Ile Phe Leu 245 250 255 Leu Leu Gly Ile Cys Trp Cys Gin Cys Cys Pro His Thr Cys Cys Cys 260 265 270 Tyr Val Arg Cys Pro Cys Cys Pro Asp Lys Cys Cys Cys Pro Giu Ala 275 280 285 Leu Tyr Ala Ala Gly Lys Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr 290 295 300 Ala Pro Ser Thr Tyr Ala His beu Ser Pro Ala Lys Thr Pro Pro Pro 305 310 315 320 Pro Ala Met Ile Pro Met Gly Pro Ala Tyr Asn Gly Tyr Pro Gly Gly 325 330 335 Tyr Pro Gly Asp Val Asp Arg Ser Ser Ser Ala Gly Gly Gin Gly Ser 340 345 350 Tyr Val Pro Leu Leu Arg Asp Thr Asp Ser Ser Val Ala Ser Giu Vai 355 360 365 Arg Ser Gly Tyr Arg Ile Gin Ala Ser Gin Gln Asp Asp Ser Met Arg 370 375 380 Val Leu Tyr Tyr Met Giu Lys Giu Leu Ala Asn Phe Asp Pro Ser Arg 385 390 395 400 Pro Gly Pro Pro Ser Gly Arg Val Giu Arg Ala Met Ser Giu Val Thr 405 410 415 Ser Leu His Giu Asp Asp Trp Arg Ser Arg Pro Ser Arg Gly Pro Ala 420 425 430 Leu Thr Pro Ile Arg Asp Giu Giu Trp Gly Gly His Scr Pro Arg Ser 435 440 445 Pro Arg Gly Trp Asp Gin Giu Pro Ala Arg Giu Gin Ala Gly Gly Gly 450 455 460 Trp Arg Ala Arg Arg Pro Arg Ala Arg Ser Val Asp Ala Leu Asp Asp 465 470 475 480 Leu Thr Pro Pro Scr Thr Ala Glu Ser Gly Scr Arg Ser Pro Thr Ser 485 490 495 Asn Gly Gly Arg Ser Arg Ala Tyr Met Pro Pro Arg Ser Arg Ser Arg 500 505 510 Asp Asp Leu Tyr Asp Gin Asp Asp Ser Arg Asp Phe Pro Arg Ser Arg 515 520 525 Asp Pro His Tyr Asp Asp Phe Arg Ser Arg Glu Arg Pro Pro Ala Asp 530 535 540 Pro Arg Ser His His His Arg Thr Arg Asp Pro Arg Asp Asfi Gly 545 550 555 560 Arg Ser Gly Asp Leu Pro Tyr Asp Gly Arg Leu Leu Giu Glu Ala Val 565 570 575 Arg Lys Lys Gly Ser Glu Glu Arg Arg Arg Pro His Lye Giu Giu Giu 580 585 590 Giu Giu Ala Tyr Tyr Pro Pro Ala Pro Pro Pro Tyr Ser Glu Thr Asp 595 600 605 Ser Gin Ala Ser Arg Glu Arg Arg Leu Lys Lys Asn Leu Ala Leu Ser 610 615 620 Arg Giu Ser Leu Val Val 625 630 <210> 6 <211> 1954 <212> DNA <213> Homo sapiens <220> <221> allele <222> 595 <223> 9-3-324 :polymorphic base C or T WO 01/21647 WO 0121647PCT/IBOO/01470 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <22 1> <222> <223> allele 987 9-7-325 allele 1158 9-9-246 33 polymorphic base A or G polymorphic base G or C allele 1454 LSRX9fl3-BM allele 1875 LSRX9f 14-EM polymorphic base deletion of polymorphic base T or G <400> 6 tggagtgtgg ctcggaggae cgcggcgggt caagcacctt atgccctttg tccacgtcgt ttacgctcat taaaacttcc tctcccceat atctgaaagc aga atg caa cag gac Met Gin Gin Asp 1 aag ggg agg age gtg Lys Gly Arg Ser Val ctt gga gta ggg Leu Gly Val Gly agg aac gga agt Arg Asn Gly Ser cac ccc tcc tgg cct tgg tgc gcg ccg cgc ccc cta agg tac ttt His Pro Ser Trp Pro Trp Cys Ala Pro Arg Pro Leu Arg Tyr Phe agg gac gcg Arg Asp Ala 9CC ggc ggg Ala Gly Gly cgg gac gcg Arg Asp Ala gee aga cgc gcc cag acg gcc geg Ala Arg Arg Ala Gin Thr Ala Ala atg gcg ctg ttg Met Ala Leu Leu etc tec aga ggg ctg ggc tee eac ccg gcc gcc gca ggc Leu Ser Arg Gly Leu Gly Ser His Pro Ala Ala Ala Gly gtc gte ttc Val Val Phe tgg ctt ctg ctt Trp, Leu Leu Leu aec tgg tge aca Thr Trp Cys Thr cct gec agg 9CC Pro Ala Arg Ala cag gtg acc gtg Gin Val Thr Val aac ccc tac cac Asn Pro Tyr His ate etc tte Ile Leu Phe ect gtg acc ctg ccc tgt ace tac cag Pro Val Thr Leu Pro Cys Thr Tyr Gin atg ace Met Thr 115 tte tgc Phe Cys teg ace ccc Ser Thr Pro egg gac ege Arg Asp Arg 135 ctc zat gee Leu Asn Ala 150 caa ccc ate gte Gin Pro Ile Val tgg aag tac aag tet Trp Lys Tyr Lys Ser gee gat gee Ala Asp Ala ttc Phe 140 99g Gly ceg gee age Pro Ala Ser 130 gte gac aac cag Val Asp Asn Gin 145 cag etg gca gce Gln Leu Ala Ala aac ec gge tac aac ccc tac gty Asn Pro Gly Tyr Asn Pro Tyr Val tge cag gac Cys Gin Asp ggc aac get gtg Gly Asn Ala Val ate ace gga aat age gtg Scr Val 170 ace ctg Thr Leu 185 get qac ege ace gte agg gte Arg Thr Val Azg Val 175 gga gat tac tac eag Gly Asp Tyr Tyr Gln 190 gee ace aag Ala Thr Lys gge egg agg Gly Arg Arg att ace Ile Thr 195 ggg gac ctg ace ttt gac cag acg geg tgg WO 01/21647 WO 0121647PCTIBOO/0 1470 Asp tgc Cys gca Al a ggt Gly 250 gcc Ala aac Asn gct Ala agt Ser cag Gin 330 aa c Asn gcc Al a cc t Pro ggc Gdy gag Giu 410 gtg Val1 agc Ser ccg Pro gac Asp -a- Giu 490 cct.

Pro cta Leu ccc Phe Asp 205 gtc tca Val Ser atc gtc Ile Val agc att Ser Ile cca ccc Pro Pro 270 cct gga Pro Gly 285 caa ggc Gin Giy tct gaa Ser Giu tcc atg Ser Met cct tct Pro Ser 350 gaa gtc Glu Val 365 ggc ccl: Gly Pro ccc cgg Pro Arg ggc ggg Gly Gly ctg gac Leu Asp 430 ccc acg Pro Thr 445 cgc agc Arg Ser cgc tcc Arg Ser cct gcc Pro Ala aac ggc Asn Gly 510 gag gct Giu Ala 525 gag gag Gin gcc Al a ctt Leu tat Tyr 255 cca Pro gga Giy tcc Se r gtc Val cgg Arg 335 cga Arg acc Thr gcc Al a agt Ser ggc- Gly 415 gac Asp agt Se r cgg Arg cgg Arg gac Asp 495 tcc Ser gtg Val1 gag Giy cag Gin Al a acc Thr at t Ile 275 gac

ASP

ctg Leu tac Tyr tac TPyr ccc Pro 355 gag Giu atc Ile tgg Trp agg Arg ccg Pro 435 aga Arg tat Tyr tac Tyr His gac Asp 515 ggg Giy tac 787 835 883 931 979 1027 1075 1123 1171 1219 1267 1315 1363 1411 1459 1507 1555 1603 1651 1699 1747 WO 01/21647 WO 0121647PCTIIBOO/01470 Glu Giu Arg Arg Arg Pro His Lys Giu Glu GiU Glu Giu Ala Tyr Tyr 535 540 545 ccg ccc gcg ccg ccc ccg tac tcg gag acc gac tcg cag gcg tcc cga Pro Pro Ala Pro Pro Pro Tyr Ser Giu Thr Asp Ser Gin Ala Ser Arg 550 555 560 gag cgc agg ctc aag aag aac ttg gcc ctg agt cgg gaa agt tta gtc Glu Arg Arg Leu Lys Lys Asn Leu Ala Leu Ser Arg Glu Ser Leu Val 565 570 575 580 gtc tga tctgacgttt tctacgtagc ttttgkattt ttttttttaa tttgaaggaa Val cactgatgaa gccctgccat acccctcccg agtctaataa aacgtataat cacaa <210> 7 <211> 581 <212> PRT c213> Homo sapiens <220> <221> VARIANT <222> 295 <223> 9-7-325 polymorphic amino acid Ser or An <220> <221> VARIANT <222> 352 <223> 9-9-246 polymorphic amino acid Pro or Arg 1795 1843 1899 1954 <220> <221> VARIANT <222> 451 <223> LSRX9f13-BM <400> 7 Met Gin Gin Asp G, 1 Gly Arg Ser Val H.

Arg Tyr Phe Gly A: Met Ala Leu Leu A: Ala Ala Ala Gly A: Thr Trp Cys Thr A:.

81 Pro Tyr His Val V 100 Tyr Gin Met Thr S4 115 Lys Ser Phe Cys Ai 130 Val Asp Asn Gin Lo 145 Asn Pro Tyr Val G' Ala Thr Lys Gin G' 180 Arg Arg Ile Thr I.' 195 Ala Trp Gly Asp S4 210 polymorphic amino acid deletion of Arg Gly Val Ser Trp Ala Arg Gly Leu Al a Val Ala Arg Leu Phe Pro Thr 120 Arg Ile 135 Ala Gin Gin Aqp Ala Val Gly Asn 200 Val Tyr 215 Leu Gin Gly Asn Asn Giu Ala Tyr Ala Glu Leu Ile Val Leu Val 230 235 240 WO 01/21647 WO 0121647PCT/IBOO/01470 Tyr Ala Ala Gly Lys Ala 245 Ala Thr Ser Leu Ser Pro 265 Pro Ala Tyr 280 Ser Ser Ser 295 Thr Asp Ser Ala Ser Gin Glu Leu Ala 345 Val Glu Arg 360 Arg Ser Arg 375 Glu Trp Gly Pro Ala Arg Ala Arg Ser 425 Glu Ser Gly 440 Tyr Met Pro 455 Asp Ser Arg Arg Ser Arg Thr Arg Asp 505 Asp Gly Arg 520 Arg Arg Arg 535 Ala Pro Pro Arg Leu Lys Gly 250 Ala Asn Ala Ser Gin 330 Asn Ala Pro Gly Glu 410 Val Ser Pro Asp Clu 490 Pro Leu Pro Pro Lys 570 <210> 8 <211> 2097 <212> DNA <213> Rattus norvegicus <400> 8 accgctcacc aggtcagttg tccccggaaa gccgaaggca tgagcttcgc ccaagttctt tttatgggtt agaactcctc cagagcgggg gaaaaaggac ttggaatagg ggcgggacgg agcacgcacc cttctccgcc ttggttcg ccgcgccccc tact'ctcggo mtacttcgga ggggacgcgc gggcaccgtc gctgctagac ggccgcg atg gcg ccg gcg gcc ggc Met Ala Pro Ala Ala Gly 1 gcg tgt gct ggg gcg cct gac tcc cac cca gct acc gtg gtc ttc gtg Ala Cys Ala Gly Ala Pro Asp Ser His Pro Ala Thr Val Val Phe Val 15 tgt ctc ttt ctc atc att ttc tgc cca gac cct gcc agt gcc atc cag Cys Leu Phe Leu Ile Ile Phe Cys Pro Asp Pro Ala Ser Ala Ile Gin -30 WO 01/21647 WO 01/ 1647PCTIBOO/01470 gig act Val Thr acc ctg Thr Leu gig tct gac ccc Val Ser Asp Pro cac gta gig aic His Val Vai Ile tic cag cca gig Phe Gin Pro Val ccc tgc acc Pro Cys Thr gig Val cag atg agc aac Gin Met Ser Asn tca tic tgc cgg Ser Phe Cys Arg aca gic ccc Thr Val Pro aic igg aag Ile Trp Lys cgi ait gcc Arg Ile Aia gat 9cc Asp Ala tic tci cci Phe Ser Pro ggc aac ccc Gly Asn Pro 105 act gic agg Thr Val Arg gtg gac aac cag cia aat gcc cag Val Asp Asn Gin Leu Asn Aia Gin tac aac ccc Tyr Asn Pro gig gag tgc cag gac Vai Glu Cys Gin Asp tig gca gct Leu Ala Ala 100 agt gta. cgc Ser Val Arg acc ctg gga Thr Leu Gly gig gig gcc Val Val Ala aaa cag ggc aat Lys Gin Gly Asn 120 gac tac Asp Tyr iac caa ggc Tyr Gin Giy aic acc ata Ile Thr Ile aat gct gac Asn Aia Asp tic gag cag Phe Glu Gin gcc igg gga gac Ala Trp Gly Asp gig tat tac Val Tyr Tyr tgc ict Cys Ser 165 gca gag Ala Glu gtg gic icg gcc Val Val Ser Ala gat cig gat Asp Leu Asp aac gag gcg Asn Giu Ala cic atc gic Leu Ile Val 185 iii cgg gcg Phe Arg Ala cit ggc agg acc Leu Gly Arg Thr gcc cct gag Ala Pro Glu cia cci ggi Leu Pro Gly ggg ccc ttg Gly Pro Leu gat tgg ctc iii Asp Trp Leu Phe gic gig gtc igc Val Val Val Cys 200 cig gcg Leu Ala agc cic cic Scr Leu Leu cic cic ctg Leu Leu Leu tgc tgg igC Cys Trp Cys igi cct cac Cys Pro His tgc tgc tat Cys Cys Tyr igi ccc igc Cys Pro Cys tgc cca Cys Pro 245 gac aag igc Asp Lys Cys acc ica ggt Thr Ser Gly 265 ica cci gcc Ser Pro Ala igc cci gag gci Cys Pro Glu Ala tat gct gct ggc Tyr Ala Ala Gly gic ccg agc aic Val Pro Ser Ile gcc ccc agc aic Ala Pro Ser Ile tat Tyr 275 at t Ile aaa gca gcc Lys Ala Ala 260 acc cac ctc Thr His Leu ccc aig ggc Pro Met Gly aag acc cca Lys Thr Pro ccg cci gcc Pro Pro Ala 280 cci ccc Pro Pro tat ggg tac Tyr Gly Tyr gac itt gac Asp Phe Asp agc ica gt Ser Ser Val 1003 1051 1099 1147 1195 1243 1291 1339 ggc cac agc icc Gly His Ser Scr ici ica gaa gia Ser 5cr Glu Val ccc cig cig Pro Leu Leu gig gat ggc Val Asp Gly agi ggc tac Ser Gly Tyr aic cag gci aac cag caa gat Ile Gin Ala Asn Gin Gin Asp 340 gac icc aig Asp Ser Met 345 gac cct icc Asp Pro Ser 360 gic cia tac Val Leu Tyr aig gag aaa gag Met Giu Lys Giu gcc aac iii Ala Asn Phe cga cci ggc Arg Pro Gly aat ggc aga Asn Gly Arg gaa cgg gcc aig Giu Arg Ala Met WO 01/21647 WO 0121647PCTIBOO/01470 gaa gta acc tcc Glu Val Thr Ser cat gaa gat gac His Glu Asp Asp cga tcg agg cct Arg Ser Arg Pro gct ect gcC Ala Pro Ala ccc atc agg Pro Ile Arg gag tgg aat Giu Trp Asn cgc cac Arg His 405 tcc cca cag Ser Pro Gin ccc aga aca tgg Pro Arg Thr Trp cag gaa ccc ctt Gin Giu Pro Leu cct cgg gcc cgc Pro Arg Ala Arg 435 caa gaa caa Gin Giu Gin 420 tct gtg gat Ser Val Asp cca agg ggt ggt tgg 9gg tct Pro Arg Gly Gly Trp Gly Ser gct cta gat gat atc aac cgg cct ggc tcc act gaa Ala Leu Asp Asp Ile Asn Arg Pro Gly Ser Thr Giu 440 445 450 tca. gga cgg tct Ser Gly Arg Ser ccc cca agt agt Pro Pro Ser Ser cgg aga gga cgg Arg Arg Gly Arg tat gca cct cca Tyr Aia Pro Pro cgc agc cgg Arg Ser Arg gat Asp 475 gat Asp ctc tat gac Leu Tyr Asp ccg Pro 480 gac Asp gac gat cct agg Asp Asp Pro Arg gac ttg Asp Leu 485 cca cat tcc Pro His Ser cca cgt gct Pro Arg Ala 505 gct ggc ttc Aia Giy Phe 520 ccc cac tat Pro His Tyr gac atc agg Asp Ile Arg tct aga gat Ser Arg Asp 500 cct cgg gat Pro Arg Asp 1387 14 1483 1531 i1579 1627 1675 1723 1771 1819 1867 1915 1963 2009 2069 2097 ccc aga tcc Pro Arg Ser cga tcc cga Arg Ser Arg agg tca agg Arg Ser Arg cag tat gat Gin Tyr Asp cga cta tta gaa Arg Leu Leu Giu gct tta aag aaa Ala Leu Lys Lys ggg tcg ggc gag aga agg agg gtt tac Gly Ser Gly Glu Arg Arg Arg Val Tyr 545 gaa gaa gag Giu Giu Glu gag ggc caa Giu Gly Gin ccc cca gca cct Pro Pro Ala Pro cca cct Pro Pro 565 aaa aag Lys Lye tac tca gag TIyr Ser Giu aat ttg gcc Asn Leu Ala 585 tcg cag gcc Ser Gin Ala gag agg agg Giu Arg Arg agt cgg gaa agt Ser Arg Giu Ser gtc gtc tga Val Val tccacgtttt gtatgtagct tttgtacttt ttttttaatt ggaatcaata ataaaatgtc taatcacaaa aaaaaaaa ttgatgaaac ttcaagccta <210> 9 <211> 593 <212> PRT <213> Rattus norvegicus <400> 9 Met Ala Pro Ala Ala Gly 1 5 Ala Thr Val Val Phe Val.

Ala Cys Ala Ala Pro Asp lie Ile Phe Cy S Pro Ala Ser Ala Ile Gin Val Ile Leu Phe Gin Pro Val Thr Thr Leu Thr Val Pro Ile Val Asp Arg Ile Ala Asp Ala Phe Leu Phe 25 Thr Val Ser His Pro Cys Pro Asp His Val Val Met Ser Asn Ser Asp Pro Leu Pro Cys Thr Ile Trp Lye Ser Pro Ala 90 Lys Ser Phe Cys Val Asp Asn Gin WO 01/21647 WOO1/1647PCTTBOO/0 1470 Gin Leu 100 Asp Ser 115 Val Thr Asn Ala Tyr Tyr Ala Tyr Leu Leu 195 Val Val Cys Trp Pro Cys Gly Lys 260 Tyr Thr 275 Ile Pro Ser Ser Asp Gly Asn Gin 340 Leu Ala 355 Giu Arg Ser Arg Trp Asn Leu Gin 420 Arg Ser 435 Ser Gly Ala Pro Pro Arg Arg Ser 500 Asp Pro Arg Leu Arg Val Ala Pro Arg Leu 580 Gly Val1 Gin Gin Ala 170 Leu Gly Leu His Cys 250 Val1 Lys Gly ser Val1 330 Arg Arg Thr Ala Ser 410 Gly Asp Ser Arg Arg 490 Asp Arg Lys Giu Thr 570 Leu Asri A-1a Arg 140 Al1a Asp Arg Leu Leu 220 Cys Pro Ser Pro Pro 300 Vali Ser Leu Gly Leu 380 Thr Arg Gly Asn dly 460 Asp Pro Axg Arg Lys 540 Glu Ser Arg Tyr 110 Lys Ile Gly Asp Ser 190 Asp Leu Cys Ala Tyr 270 Pro Asp Leu Tyr Tyr 350 Pro Glu Ile TrP Gly 430 Pro Arg Tyr Tyr Arg 510 Pro Se r dly Ala Ser 590 WO 01/21647 WO 0121647PCTIBOO/0 1470 <210> 4211> 2040 <212> DNA <213> Rattus norvegicus <400> aecgctcacc aggtcagttg tccccggaaa tttatgggtt agaactceie cagagegggg agcacgcacc etteicgee ttggttctcg ggggacgcgc gggcacegtc gctgctagae gcg igt gct ggg gcg cct gac tcc c Ala Cys Ala Gly Ala Pro Asp Ser I geegaaggca tgagcttcgc ccaagttctt gaaaaaggac ttggaatagg ggcgggacgg cegcgccecc tactctcggg atacttggga ggccgcg aig gcg ceg gcg 9CC ggc Met Ala Pro Ala Ala Gly eca get ace gtg Pro Ala Thr Val gtc tt gtg Val Phe Val gcc atc cag Ala Ile Gln tgt etc iii Cys Leu Phe gig act gig Val Thi- Val ctc ate ati tie Leu Ile Ile Phe eca gae cci gcc agi Pro Asp Pro Ala Ser tct gac ccc Ser Asp Pro eac gia gig ate His Val Val Ile tic eag cca gig Phe Gin Pro Val cig ccc tgc ace Leu Pro Cys Thicag aig age aae Gin Met Ser Asn etc aca gtc cee Leu Thr Val Pro ate tgg aag tac aag ica tic ige egg gac egi ati gcc Ile Ti-p Lys Tyr Lys Ser Phe Cys Arg Asp Arg Ile Ala gat gcc Asp Ala tt tet cei Phe Ser Pro gge aac ccc Gly Asn Pro 105 agt gig gac aac Ser Val Asp Asn eta aat gec cag Leu Asn Ala Gin tac aac ec Tryr Asn Pro gig gag tge eag Val Glu Cys Gin ttg gca get Leu Ala Ala 100 agi gia ege Ser Val Arg ace ctg gga Thi- Leu Gly act gte Thi- Val 120 gae tac Asp Tyr agg gig gtg gee ace Arg Val Val Ala Thr cag ggc aat Gin Gly Asn 120 180 235 283 331 379 427 475 523 571 619 667 715 763 811 859 907 955 1003 1051 tac caa ggc agg Tyr Gin Gly Arg 140 ate ace ata aca Ile Thr Ile Thr aat get gae Asn Ala Asp tic gag cag Phe Glu Gin gee igg gga gac Ala Trp Gly Asp gga gig tat tac Gly Val Tyr Tyr gig gte teg Val Val Scr etc ate gte Leu Ile Val 185 etc etc etc Leu Leu Leu caa gat cig gat Gin Asp Leu Asp aae aac gag geg Asn Asn Giu Ala gat tgg etc Asp Ti-p Leu gte gtg gte Val Val Val tac gca gag Tyr Ala Glu 180 cig geg age Leu Ala Ser ige igi ect Cys Cys Pro tie etc etc Phe Leu Leu ate ige tgg Ile Cys Ti-p 200 cac ace His Thr ige tge ige Cys cys Cys ega igi ccc Arg Cys Pro tge eca gac aag Cys Pro Asp Lys 215 igt Cys ige cci gag Cys Pro Glu tat get get Tyr Ala Ala gca gee ace Ala Ala Thitea ggt Ser Gly 245 gte ceg age ate Val Pro Ser Ile gee ccc age Ala Pro Ser ace cac etc Th- His Leu tea cci gee Ser Pro Ala 260 cci ccc tat Pro Pro Tyr aag ace eca cca ect ceg cci gee Lys Thr Pro Pro Pro Pro Pro Ala ati ccc atg gge Ile Pro Met Gly WO 01/21647 WOO1/1647PCT/IBOO/01470 ggg tac Gly Tyr 280 gga gac ttt Gly Asp Phe aga cat agc tca Arg His Ser Ser 275 ggt ggc cac age Gly Gly His Ser eaa gta ccc ctg Gin Val Pro Leu cgt gac gtg gat ggc agt gta tet tca Arg Asp Val Asp Giy Ser Val Ser Ser gta cga agt ggc Val Arg Ser Gly agg atc cag gct Arg Ile Gin Ala atg gag aaa gag Met Glu Lys Glu cag caa gat gac Gin Gin Asp Asp tcc atg Ser Met 325 agg gtc cta Arg Val Leu cga cet ggc Arg Pro Gly 34 1 tac Tyr 330 cct Pro gcc aac ttt Ala Asn Phe gac ect tee Asp Pro Ser 340 agt gaa gta Ser Giu Val ccc aat ggc Pro Asn Giy gtg gaa cgg gcc atg Vai Giu Arg Ala Met ace tc Thr Ser 360 etc cat gaa gat Leu His Giu Asp tgg cga tcg agg Trp Arg Ser Arg tcc agg get cct Ser Arg Aia Pro ctc ace ccc ate Leu Thr Pro Ile gat gag gag tgg Asp Giu Giu Trp cgc cac tee cca Arg His Ser Pro agt ccc aga aca Ser Pro Arg Thr cag gaa ccc Gin Giu Pro caa gaa caa cca Gin Glu Gin Pro agg ggc Arg Giy 405 ggt tgg ggg Gly Trp Gly gat atc aac Asp Ile Asn 425 agt agt gga Ser Ser Gly 440 cgc cct egg Arg Pro Arg tct gtg gat Ser Val Asp cct ggc tcc Pro Giy Ser tca gga cgg Ser Giy Arg get eta gat Ala Leu Asp 420 tct cee eca Ser Pro Pro agt cge age Ser Arg Ser 1099 11i47 1195 1243 1291 1339 1387 1435 1483 1531 1 579 1627 1675 1723 1771 1819 1867 1915 1962 2022 2040 cgg aga gga Arg Arg Gly tat gea cet Tyr Ala Pro gat gac etc tat Asp Asp Leu Tyr ccg gac gat ect Pro Asp Asp Pro gac ttg eca cat Asp Leu Pro His gat ccc cac Asp Pro His gac gac ate Asp Asp Ile aga gat cca Arg Asp Pro egt get Arg Ala 485 gac eec aga Asp Pro Arg agg tea agg Arg Ser Arg 505 aag aaa aag Lys Lys Lys cgt cag cga tee Arg Gin Arg Ser ect egg gat Pro Arg Asp cct cag tat Pro Gin Tyr ggg ega eta tta Gly Arg Leu Leu get ggc tte Ala Gly Phe 500 gag get tta Giu Ala Leu gag gaa gaa Giu Glu Giu ggg teg ggc Giy Ser Gly agg agg gtt Arg Arg Val 520 gag gaa Glu Giu gag gag ggc Glu Giu Giy gac teg eag Asp Ser Gin gee Ala 555 agt S er eaa tac ccc eca gca cet Gin Tyr Pro Pro Ala Pro 540 545 tea egg gag agg agg etg Ser Arg- Glu Arg Arg Leu 560 tta gte gte tga tccaegtt Leu Val Val cea ect tac t.

Pro Pro Tyr S aaa aag aat t Lys Lys As-n L 5I tt gtatgtagct ca gag er Giu 550 tg gee eu Ala ctg agt egg Leu Ser Arg tttgtacttt ttttttaatt ggaatcaata ttgatgaaac tteaagecta ataaaatgtc taatcacaaa aaaaaaaa <210> 11 <211> 574 WO 01/21647 WO 0121647PCT/IBOO/01470 <212> PRT <~213> Rattus norvegicus <400> 11 Met Ala Pro Ile Thr Asp Asn Cys Asn Thr 145 Gly Asn Val Trp Cys 225 Lys Thr Pro Ser Gly 305 Gin Ala Arg Arg Asn 385 Gin Ser Gly Pro Arg Ala Thr Ala Leu Leu Arg Ala Gin Ala 130 Gly Val Giu Val Cys 210 Cys Ala His Met Val 290 Ser Gin Asn Ala Pro 370 Arg Giu Val1 Arg Pro 450 Asp Ala Val1 Ala Gin Val1 Ala Leu 100 Se r Thr Ala Tyr Tyr 180 Leu Cys Asp Thr Ser 260 Pro Gly Se r Asp Asp 340 Se r Arg Se r Pro Ala 420 Se r Se r Pro Pro Ile Pro Tyr Lys Val Asn Ala Arg 140 Ala Asp Trp, Leu Tyr 220 Leu Ala Pro Phe Leu 300 Arg Met Asn Asp Arg 380 ciu Arg Gly Gly Asp 460 Tyr Ser Cys His Met Phe Asn Tyr 110 Lys Ile Gly Asp Phe 190 Gly Arg Ala Ser Ala 270 Arg Asp Gin Lys Arg 350 Trp Glu Glu Arg Thr 430 Ala Asp Asp Pro Asp Val Asn Arg Leu Giu Gly Ile Ser 160 Asn Val Cys Pro Gly 240 Tyr Ile Ser Asp Asn 320 Leu Giu Ser Trp Leu 400 Arg Ser Ala Pro Arg WO 01/21647 WO 0121647PCTIBOOIO1470 Arg Asp Pro Ala Asp Pro Arg Arg Gin Arg Ser 480 Arg Asp 495 Pro Arg Asp Leu Leu Giu 515 Val Tyr Arg Phe Arg Ser Pro Gin Tyr Asp Gly Arg 510 Arg Arg Arg Ala Leu Lys Lys Gly Ser Gly 530 Pro Pro Pro Giu Glu Glu Tyr Ser Glu Glu GlU Giu Gly Asp Ser Gin Ala Tyr Pro Pro Ala Arg Giu Arg Arg 545 Leu Lys Lys Asn Leu 565 Ala Leu Ser Arg Leu Val Val <210> 12 <211> 1893 <212> DNA <213> Rattus norvegicus <400> 12 accgctcacc t ttatgggtt agcacgcacc ggggacgcgc aggtcagttg tccccggaaa agaactcctc cagagcgggg cttetccgcc ttggttctcg gggeaccgtc gctgctagac gcegaaggca tgagcttcgc ccaagttctt gaaaaaggac ttggaatagg ggcgggacgg ccgcgccccc tactctcggg atacttggga ggccgcg atg gcg ccg gcg gee ggc Met Ala Pro Ala Ala Gly gcg tgt gct Ala Cys Ala tgt ctc ttt CYS Leu Phe gtg act gtg Val Thr Val gcg ect gac tcc Ala Pro Asp Ser cca gct ace gtg Pro Ala Thr Val gtC ttc gtg Val Phe Val gCC ate cag Ala Ile Gin etc ate att ttc Leu Ile Ile Phe gac cct gcc Asp Pro Ala tet gac ccc tac cac gta gtg ate etg ttc cag cca gtg Ser Asp Pro Tyr His Val Val Ile Leu Phe Gin Pro Val etg ccc tgc ace Leu Pro Cys Thr cag atg age aac Gin Met Ser Asn etc aca gte: ccc Leu Thr Val Pro atc tgg aag Ile Trp, Lys tea ttc tgc Ser Phe Cys cgt att gee Arg Ile Ala gat gee Asp Ala ttc tet cct gcc Phe Ser Pro Ala gtg gac aac Val Asp Asn eta aat gee cag Leu Asn Ala Gin ggc aac ccc Gly Asn Pro act gte agg Thr Val Arg 120 ggc tac aae ccc Gly Tyr Asn Pro gtg gag tge cag gac Val Glu Cys Gin Asp ttg gca get Leu Ala Ala 100 agt gta cgc Ser Val Arg ace ctg gga Thr Leu Gly gtg gtg gee Val Val Ala eag gge aat Gin Gly Asn tac tac caa ggc Tyr Tyr Gin Gly ttC gag cag aca Phe Glu Gin Thr ate ace ata Ile Thr Ile aat get gac Asn Ala Asp tgg gga gac Trp Gly Asp gtg tat tac Val Tyr Tyr tgC tct Cys Ser 165 gtg gte teg Val Val Ser etc ate gtc Leu Ile Vai 185 155 gee eaa Ala Gin 170 gat etg gat Asp Leu Asp aac aae gag geg Asn Asn Giu Ala aaa gca gcc ace Lys Ala Ala Thr 195 tac gca gag Tyr Ala Giu 180 tea ggt gte Ser Gly Val ctt gtt tat get Leu Val Tyr Ala ceg age ate tat gee ccc age ate tat ace eac etc tea ect gee aag WO 01/21647 WO 0121647PCT/IBOO/01470 Pro Ser 200 Ile Tyr Ala Pro Ile Tyr Thr His Leu Ser Pro Ala Lys cca cca cct ccg Pro Pro Pro Pro atg att CCC Met Ile Pro ggc cct ccc tat Gly Pro Pro Tyr cct gga gac Pro Gly Asp gac aga cat agc tca Asp Arg His Ser Ser ggt ggc cac Gly Gly His agc Se r 245 caa gta ccc Gin Vai Pro cga agt ggc Arg Ser Gly 265 gtc cta tac Val Leu Tyr cgt gac gtg Arg Asp Val ggc agt gta tct Gly Ser Val Ser agg atc cag Arg Ile Gin cag caa gat Gin Gin Asp tca gaa gta Ser Giu Vai 260 tcc atg agg Ser Met Arg cct tcc cga Pro Ser Arg 280 cct ggc Pro Gly cct Pro tat atg gag Tyr Met Glu ccc aat ggc Pro Asn Gly cta 9CC aac ttt Leu Ala Asn Phe gtg gaa cgg Val Giu Arg atg agt gaa gta Met Ser Glu Val ctc cat gas Leu His Giu gac tgg cga tcg Asp Trp, Arg Ser cct tcc agg gct Pro Ser Arg Ala cc t Pro 325 ctc acc ccc Leu Thr Pro ccc aga aca Pro Arg Thr 345 tgg ggg tct Trp Giy Ser 360 agg gat gag gag Arg Asp Glu Glu aat cgc cac tcc cca cag agt Asn Arg His Ser Pro Gin Ser gag cag gaa Giu Gin Giu caa gaa caa Gin Giu Gin 340 agg ggt ggt Arg Gly Giy cta gat gat Leu Asp Asp gga cgc cct Gly Arg Pro cgc tct gtg Arg Ser Val sac cgg cct ggc tcc Asn Arg Pro Gly Ser gas tca gga Giu Ser Giy tct ccc cca Ser Pro Pro 907 955 1003 1051 1099 1147 1195 1243 1291 1339 1387 1435 1483 1531 1579 1627 1675 1723 1771 1825 1885 gga cgg aga Gly Arg Arg cgg gcc tat gca Arg Ala Tyr Ala cca aga agt cgc Pro Arg Ser Arg agc cgg Ser Arg 405 gat gac ctc Asp Asp Leu gat ccc cac Asp Pro His 425 ccc aga tcc Pro Arg Ser 440 ccg gac gat Pro Asp Asp gac ttg cca Asp Leu Pro cat tcc cga His Ser Arg 420 cgt gct gac Arg Ala Asp tat gac gac Tyr Asp Asp tct aga gat cca Ser Arg Asp Pro cgt cag cga Arg Gin Arg cga gat cct cgg Arg Asp Pro Arg ggg cga cta tta Gly Arg Leu Leu gct ggc ttc agg Ala Giy Phe Arg agg gac cct cag Arg Asp Pro Gin gag gct tta.

Glu Ala Leu aag ggg tcg Lys Giy Ser gag aga agg agg GiU Arg Arg Arg gtt tac Val Tyr 480 cct cca Pro Pro agg gag gaa Arg Glu Giu gaa gag Giu Giu 485 gaa gag gag.

Glu Giu Glu gac tcg cag Asp Ser Gin 505 Ogc Gly 490 tac ccc cca Gin Tyr Pro Pro cct tac Pro Tyr tea gag act Ser Giu Thr 500 ttg 9CC ctg Leu Ala Leu 9CC tca cgg gag Ala Ser Arg Glu agg ctg aaa aag Arg Leu Lys Lys agt cgg Ser Arg 520 gsa agt tta gtc gte tga tccacgtttt gtatgtagct tttgtacttt Giu Ser Leu Val Val 525 ttttttaatt ggaatcaata ttgatgaaac ttcaagccta ataaaatgtc taatcacaaa WO 01/21647 PCT/IBOO/01470 aaaaaaaa 1893 <210> 13 <211> 525 <212> PRT <213> RattuS norvegicus <400> 13 Met Ala Pro Ala Ala Gly Ala Cys Ala Gly Ala Pro Asp Ser His Pro 1 5 10 Ala Thr Val Val Phe Val Cys Leu Phe Leu Ile Ile Phe Cys Pro Asp 25 Pro Ala Ser Ala Ile Gin Val Thr Val Ser Asp Pro Tyr His Val Val 40 Ile Leu Phe Gin Pro Val Thr Leu Pro Cys Thr Tyr Gin Met Ser Asn 55 Thr Leu Thr Val Pro Ile Val Ile Trp, Lys Tyr Lys Ser Phe Cys Arg 70 75 Asp Arg Ile Ala Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gin Leu 90 Asn Ala Gin Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val Giu 100 105 110 Cys Gin Asp Ser Val Arg Thr Val Arg Val Val Ala Thr Lys Gin Gly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gin Gly Arg Arg Ile Thr Ile 130 135 140 Thr Gly Asn Ala Asp Leu Thr Phe Glu Gin Thr Ala Trp Gly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys Ser Val Val Scr Ala Gin Asp Leu Asp Gly Asn 165 170 175 Asn Glu Ala Tyr Ala Giu Leu Ile Val Leu Val Tyr Ala Ala Gly Lys 180 185 190 Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr Ala Pro Ser Ile Tyr Thr 195 200 205 His Leu 5cr Pro Ala Lys Thr Pro Pro Pro Pro Pro Ala Met Ile Pro 210 215 220 Met Gly Pro Pro Tyr Gly Tyr Pro Gly Asp Phe Asp Arg His Ser Ser 225 230 235 240 Val Gly Gly His Ser Ser Gin Val Pro Leu Leu Arg Asp Val Asp Gly 245 250 255 Ser Vai Ser Ser Giu Val Arg Ser Gly Tyr Arg Ile Gin Ala Asn Gin 260 265 270 Gin Asp Asp Ser Met Arg Val Leu Tyr Tyr Met Giu Lys Glu Leu Ala 275 280 285 Asn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly Arg Vai Giu Arg 290 295 300 Ala Met Ser Giu Val Thr Ser Leu His Giu Asp Asp Trp, Arg Ser Arg 305 310 315 320 Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg Asp Giu Giu Trp, Asn 325 330 335 Arg His Ser Pro Gin Ser Pro Arg Thr Trp Giu Gin Glu Pro Leu Gin 340 345 350 Giu Gin Pro Arg Gly Gly Trp Gly Ser Gly Arg Pro Arg Ala Arg Ser 355, 3G0 365 Val Asp Aia Leu Asp Asp Ile Asn Arg Pro Gly Ser Thr Glu Ser Gly 370 375 380 Arg Ser Ser Pro Pro Ser Ser Gly Arg Arg Giy Arg Ala Tyr Ala Pro 385 390 395 400 Pro Arg Ser Arg Ser Arg Asp Asp Leu Tyr Asp Pro Asp Asp Pro Arg 405 410 415 Asp Leu Pro His Ser Arg Asp Pro His Tyr Tyr Asp Asp Ile Arg Ser 420 425 430 Arg Asp Pro Arg Ala Asp Pro Arg Ser Arg Gin Arg Ser Arg Asp Pro WO 01/21647 435 Arg Asp Ala 450 Leu Giu Gin 465 Tyr Arg Gin PCT/IBOO/01470 445 Asp Gly Phe Arg Ser 455 Ala Leu Lys Lys 470 Glu Giu Glu Gin Asp Pro Gin Tyr Gly Arg Leu Lys Gly Ser Glu Giu Gly 490 Arg Arg Arg Gin Tyr Pro Pro Ala Pro 495 Arg Leu Pro Pro Tyr Ser Glu Thr Asp Ser 500 Lys Lys Asfl Leu Ala Leu Ser Arg 515 520 Ala Ser Arg Giu Ser Leu Val <210> 14 <211> 1886 <212> DNA <213> Mus musculus <400> 14 gcaccgtcgc tgctagacgg ccgcg atg gcg ccg gcg gee age gcg tgt get Met Ala Pro Ala Ala Ser Ala Cys Ala ggg gcg cct ggc tce Gly Ala Pro Gly Ser ccg gcc acc acg Pro Ala Thr Thr gac cgt gce agt Asp Arg Ala Ser ttc gtg tgt ctt ttt Phe Val Cys Leu Phe atc att tac Ile Ile Tyr atc cag gtg Ile Gin Val acc gtg Thr Val cct gac ccc Pro Asp Pro tgc acc tac Cys Thr Tyr gia gtg atc Val Val Ile cag eca Gin Pro atg agc aat Met Ser Asn aca gcc ect.

Thr Ala Pro gtg aca cta cac Val Thr Leu His atc gtg ate tgg Ile Val Ile Trp gcc ttc tcc cet Ala Phe Ser Pro aag tat Lys Tyr aag tcg ttc tgt Lys Ser Phe Cys gac egt gtt gcc Asp Arg Val Ala gac Asp gcg Al a gcc Ala age gtg gac aac eag etc aac gec cag Ser Val Asp Asn Gin Len Asn Ala Gin gct gge aac Ala Gly Asn ggc tac aac ccc Gly Tyr Asn Pro gtg gtg gcc acc Val Val Ala Thr gtg gag tgc cag Val Giu Cys Gin cag ggc aat get.

Gin Gly Asn Ala gta cgc act Val Axg Thr gte agg Val1 Arg 120 acc ctg gga Thr Leu Gly gac tac tac Asp Tyr Tyr 135 acc ttc gag Thr Phe Giu cag ggc agg Gin Gly Arg 140 cag acg gc Gin Thr Ala ate acc atc aca Ile Thr Ile Thr aat get ggc Asn Ala Gly tgg gga gae Trp, Giy Asp gga gtg tat tac Gly Val Tyr Tyr tcc gtg gte tca Ser Val Val Ser 155 gce caa Ala Gin gat ctg gat Asp Leu Asp aac aac gag gcg Asn Asn Glu Ala gag etc att.

Glu Leu Ile ggc agg acc tea gaa gcc cct gag Gly Arg Thr Ser Giu Ala Pro Gin cta cct. ggt. ttt Leu Pro Gly Phe cgg gcg Arg Ala 200 ggg ccc ttg Gly Pro Leu etc etc tte Len Len Phe 220 gaa Giu 205 gat tgg etc ttt gtg Asp Trp Len Phe Val gtg gte tge Val Val Cys ctg gca agc Leu Ala Ser 215 tgc tgt ccc Cys Cys Pro ttc ctc ctc ctg Phe Leu Leu Leu ate tge tgg tgc Ile Cys Trp, Cys eag Gin 230 WO 01/21647 WO 0121647PCT/LBOO/01470 cac acc His Thr 235 igt tgc Cys Cys 250 tgc tgc igc tat Cys Cys Cys Tyr aga igt ccc tgc Arg Cys Pro Cys cca gac aag tgC Pro Asp Lys Cys cct gag gcc: Pro Glu Ala gct gci ggc Ala Ala Gly 9CC acc tca Ala Thr Ser gig cca agc aic Val Pro ser Ile ccc agc atc Pro Ser Ile cac cic tct His Leu Ser cct gcc Pro Ala 280 aag act ccg Lys Thr Pro ggg tac cct Gly Tyr Pro 300 tcc cag gtg Ser Gin Val ccg cct gce Pro Pro Ala att ccc atg cgi Ile Pro Met Arg gga gac: Lit gac Gly Asp Phe Asp agc ica gt Scr Ser Val cct ccc: tat Pro Pro Tyr 295 ggc cac agc Giy His Ser ici tca gaa Ser Ser Giu ccc ctg ctg Pro Leu Leu gig gat ggg Vai Asp Giy 315 gia cga Vai Arg agt ggc tac Ser Giy Tyr cag gci aac Gin Ala Asn gat gac icc Asp Asp Ser gtc cia tac Vai Leu Tyr gag aag gag cia Glu Lys Giu Leu aac tic gai Asn Phe Asp cci icc Pro Ser 360 gaa gia Glu Val cgg cci ggc Arg Pro Gly acc icc cic Thr Ser Leu 380 aai ggc ega Asn Gly Axg gaa cgg gcc aig Giu Arg Ala Met gaa gat gac Giu Asp Asp cga tct Cgg ccL Arg Ser Arg Pro agg gci ccL Arg Ala Pro icc ci Cgg Ser Pro Arg gcc ctc Ala Leu 395 agi ccc Ser Pro aca ccc aic agg Thr Pro Ile Arg gag igg aai Glu Trp, Asn 772 820 868 916 964 1012 1060 1108 1156 1204 1252 1300 1348 1396 1444 1492 1540 1588 1636 1684 1732 aga aca igg Arg Thr Trp, gaa ccc cii caa Giu Pro Leu Gin cag cca agg Gin Pro Arg igg 999 ict Trp Gly Ser ci Cgg cC Pro Arg Ala ict gig gat gct Ser Vai Asp Ala cta gat Leu Asp 440 gac aLC aac Asp Ile Asn agi agt gga Ser Ser Giy 460 cgg gat gac Arg Asp Asp cci ggc Lcc aci Pro Giy Ser Thr ica gga agg Lci Ser Gly Arg Ser ict ccc cca Ser Pro Pro 455 agi cgc agc Ser Arg Ser cgg aga ggg cgg gcc tat gca cct ccg Arg Arg Giy Arg Aia Tyr Ala Pro Pro 465 cic tat gac ccc gac gaL cct aga gac Leu Tyr Asp Pro Asp Asp Pro Arg Asp tig cca cat icc Leu Pro His Ser 475 cga gat Arg Asp ccc cac tat Pro His Tyr gat gat tig agg Asp Asp Leu Arg gat cca cgt Asp Pro Arg ccc aga icc Pro Arg Ser cga icc cac Arg Ser His cgg gai get Arg Asp Ala ggc tic Giy Phe 520 agg ica cgg Arg Ser Arg aag aaa aaa Lys Lys Lys 540 gaa gaa gaa Glu Giu Glu 555 cci cag tat gat Pro Gin Tyr Asp ctc ita gaa Leu Leu Glu gct 999 gag Ala Gly Glu cgc gtt tac Arg Vai Tyr gag gct ita Glu Aia Leu 535 gag gaa gaa Giu Giu Giu cci tac ici Pro Tyr Ser gag gag ggc Glu Glu Gly CCC cca gca Pro Pro Ala WO 01/21647 WO 0121647PCT/IBOO/01470 act gac tcg Thr Asp Ser eag 9cc teg agg gag cgg agg Gin Ala Ser Arg Giu Arg Arg 575 580 atg aaa aag aat ttg Met Lys Lys Asn Leu 585 gcc ctg agt cgg gaa agt tta gtc gic tga tcccacgttt tgtiatgtag Ala Leu Ser Arg Glu Ser Leu Val Val 590 595 cttttatact titttaattg gaatattgat gaaaecctc accaagccta ataaaa <210> <211> 1829 <212> DNA <213> Mus musculus 1780 1830 1886 <400> gcaccgtcgc tgctagacgg ccgcg atg gcg ccg gcg 9CC agc gcg tgt get Met Ala Pro Ala Ala Ser Ala Cys Ala gcg cct ggc tcc cac ccg gcc acc acg Ala Pro Gly Ser His Pro Ala Thr Thr ttc gig tgi ctt Phe Val Cys Leu atc cag gtg acc Ile Gin Val Thr ate ati tac Ile Ilie Tyr cca gac egi gec agt Pro Asp Arg Ala Ser t ii Phe gtg Val cac His cci gac ccc tac Pro Asp Pro Tyr gta gig atc Val Val Ile ttc cag cca gig Phe Gin Pro Val cia Leu ige ace tac eag atg agc aat acc Cys Thr Tyr Gin Met Ser Asn Thr ctc aca gcc cci Leu Thr Ala Pro gtg atc igg Val Ile Trp aag tat Lys Tyr 9CC agc Ala Ser aag tcg tic igi Lys Ser Phe Cys gac cgt git gcc gac gcc tic ice cci Asp Arg Vai Ala Asp Aia Phe Ser Pro gtg gac aac cag etc aae gcc cag etg Val Asp Asn Gin Leu Asn Ala Gin Leu get ggc aae Ala Gly Asn tac aac ccc Tyr Asn Pro gig gag igc cag gac Val Glu Cys Gin Asp gia cgc act Val Arg Thr gtg gig gee ace Val Val Ala Thr 125 cag gge aat Gin Giy Asn gig ace etg gga Val Thr Leu Gly cag ggc agg Gin Gly Arg 140 cag aeg gee Gin Thr Ala aga ate acc aic aca gga aat get gge Arg Ilie Thr Ilie Thr Gly Asn Ala Gly gac inc tac Asp Tyr Tyr 135 ace tic gag Thr Phe Glu gig gte tea Val Val Ser tgg gga gae Trp Gly Asp gga gtg tat tac Gly Val Tyr Tyr 155 gee eaa Ala Gin gat ctg gat Asp Leu Asp aae gag geg Asn Glu Ala gag ete att Glu Leu Ile gat igg etc Asp Trp Leu gig gte gtg gte Val Val Val Val gca agc cic Ala Ser Leu tie etc ete Phe Leu Leu ige tgc tat Cys Cys Tyr 220 gag gee cii GiU Ala Leu ate tge tg Ile Cys Trp tge tgt ec Cys Cys Pro Cac ace ige His Thr Cys 215 tgi tge cci

C

1 's Cys Pro aga tgt ccc Arg Cys Pro tge Cys 225 aaa Lye eca gac aag Pro Asp Lys tat get gci Tyr Ala Ala gca gee ace Ala Ala Thr 235 ate tat ggi gig cen age Gly Vai Pro Ser gee aag act ceg gee ccc age ate tat ace cac etc tet WO 01/21647 WO 0121647PCT/IBOO/0 1470 Ile Tyr Ala Pro Ser 250 cca cct ccg ect gee Pro Pro Pro Pro Ala Tyr Thr His Leu Pro Ala Lys Thr att ccc atg Ile Pro Met cet ccc tat ggg Pro Pro Tyr Gly tac ect Tyr Pro 280 gga gac ttt Gly Asp Phe eec etg ctg Pro Leu LeU agg acc age tca Arg Thr Ser Ser ggt gge cac agc Gly Gly His Ser gaa gtg gat Glu Val Asp ggg Gly 305 cag Gin age gta tet tca Ser Val Ser Ser tcc eag gtg Ser Gin Val 295 gta ega agt Val Arg Ser agg gte eta Arg Vai Leu gge tae Giy Tyr 315 tae tat Tyr Tyr agg ate eag get aae Arg Ile Gin Ala Asn eaa gat gae Gin Asp Asp atg gag aag Met Giu Lys eta gee aae ttc gat tLeu Ala Asn Phe Asp tec egg eeL Ser Arg Pro eet eec aat gge Pro Pro Asn Gly ega.

Arg 350 tgg Trp, gtg gaa egg gee Val Glu Arg Ala agt gaa gta ace Ser Glu Val Thr eat gaa gat His Giu Asp eec ate agg Pro Ile Arg 380 aca tgg gag Thr Trp, Glu ega tet egg Arg Ser Arg tce agg get eeL Ser Arg Ala Pro gee etc aca Ala Leu Thr 375 agt eec aga Ser Pro Arg gat gag gag tgg Asp Glu Giu Trp ege eae tee eeL Arg His Ser Pro 395 Let ggg Ser Gly egg Arg eag gaa c Gin Giu Pro eeL egg gee Pro Arg Ala gaa eag eca Glu Gin Pro ggt ggt tgg ggg Gly Giy Trp Gly tet gtg gat Ser Val Asp eta gat gae ate Leu Asp Asp Ile eeL gge tee Pro Giy Ser tea gga agg Ser Gly Arg eec eca agt Pro Pro Ser agt gga Ser Gly 440 gaL gac Asp Asp egg aga ggg Arg Arg Gly etc tat gac Leu Tyr Asp 460 eae tat tat His Tyr Tyr gee tat gea eet Ala Tyr Ala Pro aga agt ege age Arg Ser Arg Ser 1012 1060 1108 1156 1204 1252 1300 1348 1396 1444 1492 1540 1588 1636 1684 1732 1783 1829 cee gae gat ect Pro Asp Asp Pro ttg eca eat Leu Pro His ega gaL ec Arg Asp Pro gae eec aga Asp Pro Arg gaL gat ttg Asp Asp Leu agg Let agg Arg Ser Arg 480 gaL eea Asp Pro 475 Lee cgt Ser Arg eag ega tee Gin Arg 5cr gat Asp ega Arg eeL cag tat gat Pro Gin Tyr Asp eet egg gat get Pro Arg Asp Ala 500 etc tta gaa gag Leu Leu Glu Giu 515 gtt Lae agg gag Val Tyr Arg Giu tte agg tea Phe Arg Ser get Lta aag Ala Leu Lys aaa aaa Lys Lys 520 ggg get ggg Gly Ala Gly gag gag a~c Giu Giu Giy 540 aga ege Arg Arg gaa gaa Giu Glu tat ccc eea Tyr Pro Pro eeL ceg cct tac Pro Pro Pro Tyr gaa gaa gaa Glu Giu Giu 535 gag act gac Giu Thr Asp gee cig agt Ala Leu Ser Leg eag Ser Gin 555 gee Leg agg gag Ala Ser Arg Giu atg aaa aag Met Lys Lys gaa agt tta gte Glu Ser Leu Val tga teeeacgttt tgttatgtag cttttatact tttttaattg gaatattgat gaaaetette aeeaageeta ataaaa WO 01/21647 WO 0121647PCTIiBOO/01470 <210> 16 <211> 16B2 <212> DNA <213> Mus musculus <400> 16 gcaccgtcgc tgctagacgg ccgcg atg gcg ccg gcg gcc agc gcg tgt gct Met Ala Pro Ala Ala Ser Ala Cys Ala ggg gcg cct ggc tce Gly Ala Pro Gly Ser ccg 9CC acc acg atc ttc gtg tgt ett ttt Pro Ala Thr Thr Ile Phe Val Cys Leu Phe ctc ate att Leu Ile Ile ect gac ccc Pro Asp Pro tge acc tac Cys Thr Tyr aag tat aag Lys Tyr Lys tac tge Tyr Cys tac cac Tyr His cea gac cgt gcc Pro Asp Arg Ala gee ate cag gtg Ala Ile Gin Val acc gtg Thr Val gta gtg atc Val Val Ile ttc cag cca gtg Phe Gin Pro Val eag atg agc aat Gin Met Ser An aca gCC ect Thr Ala Pro aca cta cac Thr Leu His gtg ate tgg Val Ile Trp tte tcc ect Phe Ser Pro gcc age Ala Ser gtg Val1 tcg tte tgt Ser Phe Cys gac aac eag Asp Asn Gin egt gtt gc Arg Vai Ala aac gee eag Asn Ala Gin get gge aae Ala Gly An tac aae eec tat gtg gag tge eag gae age gta ege act Tyr Asn Pro Tyr Val Giu Cys Gin Asp Ser Val Arg Thr gte agg Val1 Arg 120 110 115 gtg gtg 9CC Val Val Ala cag ggc agg Gin Giy Arg 140 cag aeg gee Gin Thr Ala aaa eag gge aat get gtg ace etg gga Lys Gin Gly Ann Ala Val Thr Leu Gly ate ace ate aca Ile Thr Ile Thr aat gct ggc Asn Ala Giy gac tac tac Asp Tyr Tyr 135 ace tte gag Thr Phe Giu gtg gte tea Val Val Ser tgg gga gac agt gga gtg tat tac Trp Gly Asp Ser Gly Val Tyr Tyr 155 gee caa Ala Gin gat ctg gat Asp Leu Asp aae aac gag geg tac Asn Ann Giu Ala Tyr gag etc att Giu Leu Ile gtt tat get Val Tyr Ala aaa gca gee Lys Ala Ala ggt gtg eea age Gly Val Pro Ser 200 tat gee ccc Tyr Ala Pro ect eeg cet Pro Pro Pro 220 gac ttt gac Asp Phe A~n ate tat ace cac Ile Tyr Thr His tet ect gee aag Ser Pro Ala Lys act ceg eca Thr Pro Pro 215 tae ect gga Tyr Pro Gly atg att c Met Ile Pro ect eec tat Pro Pro Tyr agg ace age tea gtt ggt gge cac age tee cag gtg ec Arg Thr Ser Ser Val Gly Gly His S9er Ser Girt Val Pro 240 245 235 etg etg Leu Leu 676 724 772 820 868 916 egt gaa gtg Arg Giu Val gat Asp 255 ggg age gta tet Gly Ser Val Ser gaa gta cga agt Giu Val Arg Ser 250 t ae Tyr agg atc cag get aae eag caa gat gac Arg Ile Gin Ala Asn Gin Gin Asp Asp 270 275 tee atg agg gte Ser Met Arg Val eta tac Leu Tyr 280 gge cet Gly Pro tat atg gag aag gag eta gee aac tte gat cet tee egg eet Tyr Met Glu Lys Giu Leu Ala Asn Phe Asp Pro Ser Arg Pro WO 01/21647 cec aat gge Pro Asn Gly 300 PCTIBOOO1470 285 cga gtg gaa cgg Arg Val Giu Arg atg agt gaa gta Met Ser Giu Val 295 tcc ctc cat Ser Leu His gaa gat Giu Asp 315 atc agg Ile Arg gac tgg cga tct ASP Trp Arg Ser cct tcc agg gct Pro Ser Arg Ala gcc etc aca ccc Ala Leu Thr Pro gat gag gag Asp Glu Glu aat cgc cac tee Asn Arg His Ser agt ccc aga Ser Pro Arg gag cag gaa Glu Gin Glu caa gaa cag Gin Glu Gin ggt ggt tgg Giy Gly T-p, ggg C99 eeL cgg Gly Arg Pro Arg cgc tet gtg Arg Ser Val cta gat gac Leu Asp Asp ate aac egg Ile Asn Arg 375 agt gga egg Ser Giy Arg cct ggc tcC Pro Gly Ser 380 act gaa tca gga Thr Giu Ser Gly tet tet ccc cea Ser Ser Pro Pro aga ggg Arg Gly 395 tat gac Tyr Asp cgg gec tat gca cet Arg Ala Tyr Ala Pro aga agt cgc Arg Ser Arg cgg gat gac etc Arg Asp Asp Leu ccc gac gat Pro Asp Asp aga gac ttg eca Arg Asp Leu Pro tee cga gat c Ser Arg Asp Pro 964 1012 1060 1108 1156 1204 1252 1300 1348 1396 1444 1492 1540 1588 1636 1682 tat gat gat Tyr Asp Asp tet agg gat Ser Arg Asp get gae ccc Ala Asp Pro aga. tc Arg Ser 440 egt eag ega.

Arg Gin Arg eeL eag tat Pro Gin Tyr 460 tee Ser 445 gat Asp gat cet egg gat Asp Pro Arg Asp gge tte agg Gly Phe Arg tea egg gac Ser Arg Asp 455 aaa aaa ggg Lys Lys Gly ggg cga etc Gly Arg Leu gaa. gag get tta Glu Glu Ala Leu get ggg Ala Gly 475 gag gge Giu Gly gag aga aga ege Glu Arg Arg Arg tac agg gag gaa Tyr Arg Giu Glu gaa gaa gaa gag Giu Glu Glu Glu cac tat ccc His Tyr Pro ect ceg eeL Pro Pro Pro gag act gac Giu Thr Asp 490 cag Gin gee teg agg Ala Ser Arg agg atg aaa Arg Met Lys aat ttg gee etg Asn Leu Ala Leu agt Ser 520 gaa agt tta Glu Ser Leu tga teccacgttt tgttatgtag cttttataet tttttaattg gaatattgat gaaactcttc aceaagccta ataaaa <210> 17 <211> 594 <212> PRT <213> Mus musculus -400> 17 Met Ala Pro Ala Ala Ser Ala Cys Ala Gly Ala Pro Gly Ser His Pro 1 5 10 Ala Thr Thr Ile Phe Val Cys Leu Phe Leu Ile Ile Tyr Cys Pro Asp 25 Arg Ala Ser Ala Ile Gin Val Thr Val Pro Asp Pro Tyr His Val Val 40 Ile Leu Phe Gin Pro Val Thr LeU His Cys Thi- Tyr Gin Met Ser Asn so 55 Thi- Leu Thr Ala Pro Ilie Val Ile Ti-p Lys Tyr Lys Ser Phe Cys Arg WO1/21647 PCT/IBOO/01470 52 70 75 Asp Arg Val Ala Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gin Leu 90 Asn Ala Gin Leu Ala Ala Giy Asn Pro Gly Tyr Asn Pro Tyr Val Glu 100 105 110 Cys Gin Asp Ser Val Arg Thr Val Arg Val Val Ala Thr Lys Gin Gly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gin Gly Arg Arg Ile Thr Ile 130 135 140 Thr Gly Asn Ala Gly Leu Thr Phe Giu Gin Thr Ala Trp Gly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gin Asp Leu Asp Gly Asn 165 170 175 Asn Giu Ala Tyr Ala Glu Leu Ile Val Leu Gly Arg Thr Ser Glu Ala 180 185 190 Pro Glu Leu Leu Pro Gly Phe Arg Ala Gly Pro Leu Glu Asp Trp Leu 195 200 205 Phe Val Val Val Val Cys Leu Ala Ser Leu Leu Phe Phe Leu Leu Leu 210 215 220 Gly Ile Cys Trp Cys Gin Cys Cys Pro His Thr Cys Cys Cys Tyr Val 225 230 235 240 Arg Cys Pro Cys Cys Pro Asp Lys Cys Cys Cys Pro Glu Ala Leu Tyr 245 250 255 Ala Ala Gly Lys Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr Ala Pro 260 265 270 Ser Ilie Tyr Thr His Leu Ser Pro Ala Lys Thr Pro Pro Pro Pro Pro 275 280 285 Ala Met Ile Pro Met Arg Pro Pro Tyr Gly Tyr Pro Gly Asp Phe Asp 290 295 300 Arg Thr Ser Ser Val Gly Gly His Ser Ser Gin Val Pro Leu Leu Arg 305 310 315 320 Glu Val Asp Gly Ser Val Ser Ser Glu Val Arg Ser Gly Tyr Arg Ile 325 330 335 Gin Ala Asn Gin Gin Asp Asp Ser Met Arg Val Leu Tyr Tyr Met Giu 340 345 350 Lys Giu Leu Ala Asn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly 355 360 365 Arg Val Giu Arg Ala Met Ser Glu Val Thr Ser Leu His Giu Asp Asp 370 375 380 Trp Arg Ser Arg Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg Asp 385 390 395 400 Giu Glu Trp Asn Arg His Ser Pro Arg Ser Pro Arg Thr Trp Giu Gin 405 410 415 Giu Pro Leu Gin Giu Gin Pro Arg Gly Gly Trp Gly Ser Gly Arg Pro 420 425 430 Arg Ala Arg Ser Val Asp Ala Leu Asp Asp Ile Asn Arg Pro Gly Ser 435 440 445 Thr Giu Ser Gly Arg Ser Ser Pro Pro Ser Ser Gly Arg Arg Gly Arg 450 455 460 Ala Tyr Ala Pro Pro Arg Ser Arg Ser Arg Asp Asp Leu Tyr Asp Pro 465 470 475 480 Asp Asp Pro Arg Asp Leu Pro His Ser Arg Asp Pro His Tyr Tyr Asp 485 490 495 Asp Leu Aro Ser Ara Asp Pro Ara Ala Asp Pro Arg Ser Arg Gin Arg 500 505 510 Ser His Asp Pro Arg Asp Ala Gly Phe Arg Ser Arg Asp Pro Gin Tyr 515 520 525 Asp Gly Arg Leu Leu Giu Giu Ala Leu Lys Lys Lys Gly Ala Gly Giu 530 535 540 Arg Arg Arg Val Tyr Arg Giu dlii Glu Glu Glu Glu Glu Glu Gly His 545 550 555 560 Tyr Pro Pro Ala Pro Pro Pro Tyr Ser dlu Thr Asp Ser Gin Ala Ser 565 570 575 WO 01/21647 PCTIBOO/01470 53 Arg Glu Arg Arg Met Lys Lys Asn Leu Ala Leu Ser Arg Glu Ser Leu 580 585 590 Val Val <210> 18 <211> 575 <212> PRT <213> Mus musculus <400> 18 Met Ala Pro Ala Ala Ser Ala Cys Ala Gly Ala Pro Gly Ser His Pro 1 5 10 Ala Thr Thr Ilie Phe Val Cys Leu Phe Leu Ile Ile Tyr Cys Pro Asp 25 Arg Ala Ser Ala Ile Gin Val Thr Val Pro Asp Pro Tyr His Val Val 40 Ile Leu Phe Gin Pro Val Thr Leu His Cys Thr Tyr Gin met Ser Asn 55 Thr Leu Thr Ala Pro Ile Val Ile Trp Lys Tyr Lys Ser Phe Cys Arg 70 75 s0 Asp Arg Val Ala Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gin Leu 90 Asn Ala Gin Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val Glu 100 105 110 Cys Gin Asp Ser Val Arg Thr Val Arg Val Val Ala Thr Lys Gin Gly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gin Gly Arg Arg Ile Thr Ile 130 135 140 Thr Gly Asn Ala Gly Leu Thr Phe Glu Gin Thr Ala Trp Gly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gin Asp Leu Asp Gly Asn 165 170 175 Asn Glu Ala Tyr Ala Giu Leu Ile Val Leu Asp Trp Leu Phe Val Vai 180 185 190 Val Val Cys Leu Ala Ser Leu Leu Phe Phe Leu Leu Leu Gly Ile Cys 195 200 205 Trp Cys Gin Cys Cys Pro His Thr Cys Cys Cys Tyr Val Arg Cys Pro 210 215 220 Cys Cys Pro Asp Lys Cys Cys Cys Pro Giu Ala Leu Tyr Ala Ala Gly 225 230 235 240 Lys Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr Ala Pro Ser Ile Tyr 245 250 255 Thr His Leu Ser Pro Ala Lys Thr Pro Pro Pro Pro Pro Ala Met Ile 260 265 270 Pro Met Arg Pro Pro Tyr Gly Tyr Pro Gly Asp Phe Asp Arg Thr Ser 275 280 285 Ser Val Gly Gly His Ser Ser Gin Val Pro Leu Leu Arg Glu Val Asp 290 295 300 Giy Ser Val Ser Ser Glu Val Arg Ser Gly Tyr Arg Ile Gin Ala Asn 305 310 315 320 Gin Gin Asp Asp Ser Met Arg Val Leu Tyr Tyr Met Giu Lys Giu Leu 325 330 335 Ala Asn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly Arg Val Giu 340 345 350 Arg Ala Met Ser Giu Val Thr Ser Leu His Glu Asp Asp Trp Arg Ser 355 360 365 Arg Pro Ser Arg Ala Pro Ala Leu Thr Pro Ile Arg Asp Giu Giu Trp, 370 375 380 Asn Arg His Ser Pro Arg Ser Pro Arg Thr Trp Glu Gin Giu Pro Leu 385 390 395 400 Gin Glu Gin Pro Arg Gly Gly Trp Gly Ser Gly Arg Pro Arg Ala Arg 405 410 415 Ser Val Asp Ala Leu Asp Asp Ile Asn Arg Pro Gly Ser Thr Giu Ser WO 01/21647 PCT/IB00/01470 54 420 425 430 Gly Arg Ser Ser Pro Pro Ser Ser Gly Arg Arg Gly Arg Ala Tyr Ala 435 440 445 Pro Pro Arg Ser Arg Ser Arg Asp Asp Leu Tyr Asp Pro Asp Asp Pro 450 455 460 Arg Asp Leu Pro His Ser Arg Asp Pro His Tyr Tyr Asp Asp Leu Arg 465 470 475 480 Ser Arg Asp Pro Arg Ala Asp Pro Arg Ser Arg Gin Arg Ser His Asp 485 490 495 Pro Arg Asp Ala Gly Phe Arg Ser Arg Asp Pro Gln Tyr Asp Gly Arg 500 505 510 Leu Leu Glu Glu Ala Leu Lys Lys Lys Gly Ala Gly Glu Arg Arg Arg 515 520 525 Val Tyr Arg Glu Glu Glu Glu Glu Glu Glu Glu Gly His Tyr Pro Pro 530 535 540 Ala Pro Pro Pro Tyr Ser Glu Thr Asp Ser Gin Ala Ser Arg Glu Arg 545 550 555 560 Arg Met Lys Lys Asn Leu Ala Leu Ser Arg Glu Ser Leu Val Val 565 570 575 <210> 19 <211> 526 <212> PRT <213> Mus musculus <400> 19 Met Ala Pro Ala Ala Ser Ala Cys Ala Gly Ala Pro Gly Ser His Pro 1 5 10 Ala Thr Thr Ile Phe Val Cys Leu Phe Leu Ile Ile Tyr Cys Pro Asp 25 Arg Ala Ser Ala Ile Gin Val Thr Val Pro Asp Pro Tyr His Val Val 40 Ile Leu Phe Gin Pro Val Thr Leu His Cys Thr Tyr Gln Met Ser Asn 55 Thr Leu Thr Ala Pro Ile Val Ile Trp Lys Tyr Lys Ser Phe Cys Arg 70 75 Asp Arg Val Ala Asp Ala Phe Ser Pro Ala Ser Val Asp Asn Gin Leu 90 Asn Ala Gin Leu Ala Ala Gly Asn Pro Gly Tyr Asn Pro Tyr Val Glu 100 105 110 Cys Gln Asp Ser Val Arg Thr Val Arg Val Val Ala Thr Lys Gin Gly 115 120 125 Asn Ala Val Thr Leu Gly Asp Tyr Tyr Gln Gly Arg Arg Ile Thr Ile 130 135 140 Thr Gly Asn Ala Gly Leu Thr Phe Glu Gin Thr Ala Trp Gly Asp Ser 145 150 155 160 Gly Val Tyr Tyr Cys Ser Val Val Ser Ala Gin Asp Leu Asp Gly Asn 165 170 175 Asn Glu Ala Tyr Ala Glu Leu Ile Val Leu Val Tyr Ala Ala Gly Lys 180 185 190 Ala Ala Thr Ser Gly Val Pro Ser Ile Tyr Ala Pro Ser Ile Tyr Thr 195 200 205 His Leu Ser Pro Ala Lys Thr Pro Pro Pro Pro Pro Ala Met Ile Pro 210 215 220 Met Arg Pro Pro Tyr Gly Tyr Pro Gly Asp Phe Asp Arg Thr Ser Ser 225 230 235 240 Val Gly Gly His Ser Ser Gin Val Pro Leu Leu Arg Glu Val Asp Gly 245 250 255 Ser Val Ser Ser Glu Val Arg Ser Gly Tyr Arg Ile Gin Ala Asn Gin 260 265 270 Gin Asp Asp Ser Met Arg Val Leu Tyr Tyr Met Glu Lys Glu Leu Ala 275 280 285 Asn Phe Asp Pro Ser Arg Pro Gly Pro Pro Asn Gly Arg Val Glu Arg WO 01/21647 PTIO/17 PCT/IBOO/01470 Arg Glu Pro 350 Al a Glu Tyr Asp Leu 430 His Gly Arg Pro Glu 510 Val1 <210> <211> 18 <212> DNA <213> Homo Sapiens <220> <221> misc -binding <222> 1-.18 <223> sequencing oligonucleotide PrimerPJ <400> tgtaaaacga cggccagt <210> 21 <211> 18 <212> DNA <213> Homo Sapiens <220> <221> misc -binding <222> 18 <223> sequencing oligonucleotide PrimerRP <400> 23 caggaaacag ctatgacc <210> 22 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide sense primer WO 01/21647 PCT/IBOO/01470 56 <400> 22 ctacaacccc tacgtcgagt <210> 23 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide anti sense primer <400> 23 aggcggagat cgccagtcgt <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide sense primer <400> 24 cctttgtcca cgtcgtttac gctc 24 <210> <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide anti sense primer <400> tcacagcgtt gccctgcttg <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide sense primer <400> 26 ttactgctcc gtggtctcag c 21 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence ,:!220> <223> oligonucleotide anti sense primer <400> 27 agctactcct gtcaacgtct cc 22 <210> 28 <211> 167 <212> PRT <213> Bos taurus WO 01/21647 PCT/IBOO/01470 57 <400> 28 Met Arg Cys Gly Pro Leu Tyr Arg Phe Leu Trp Leu Trp Pro Tyr Leu 1 5 10 Ser Tyr Val Giu Ala Val Pro Ile Arg Lys Val Gin Asp Asp Thr Lys 25 Thr Leu Ile Lye Thr Ile Val Thr Arg Ile Asn. Asp Ile Ser His Thr 40 Gin Ser Val Ser Ser Lys Gin Arg Vai Thr Giy Leu Asp Phe Ile Pro 55 Gly Leu His Pro Leu Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala 70 75 Ile Tyr Gin Gin Ile Leu Thr Ser Leu Pro Ser Axg Asn Val Vai Gin 90 Ile Ser Asn Asp Leu Giu Asn Leu Arg Asp Leu Leu His Leu Leu Aia 100 105 110 Ala Ser Lys Ser Cys Pro Leu Pro Gin Val Arg Ala Leu Glu Ser Leu 115 120 125 Giu Ser Leu Gly Val Val Leu Glu Ala Ser Leu Tyr Ser Thr Giu Val 130 135 140 Val Ala Leu Ser Arg Leu Gin Giy Ser Leu Gin Asp Met Leu Arg Gin 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys 165 <210> 29 <211> 146 <212> PRT <213> Canis familiaris <400> 29 Val Pro Ile Arg Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ilie Vai Ala Arg Ile Asn Asp Ile Ser His Thr Gin Ser Val Ser Ser 25 Lye Gin Arg Val Ala Gly Leu Asp Phe Ile Pro Gly Leu Gin Pro Val 40 Leu Ser Leu Ser Arg Met Asp Gin Thr Leu Ala Ile Tyr Gin Gin Ile 55 Leu Asn Ser Leu His Scr Arg Aen Val Val Gin Ile Ser Asn Asp Leu 70 75 Glu Aen Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lye 5cr Cys 90 Pro Leu Pro Arg Ala Arg Gly Leu Giu Thr Phe Glu Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Giu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Ala Ala Leu Gin Asp Met Leu Arg Arg Leu Asp Leu Ser Pro 130 135 140 Giy Cys 145 <210> <2i1> 163 <212> PRT <213> Galius galius <400> Met Cys Trp Arg Pro Leu Cys Arg Leu Trp, Ser Tyr Leu Val Tyr Val 1 5 10 Gin Ala Val Pro Cys Gin Ilie Phe Gin Asp Asp Thr Lye Thr Leu Ile 25 Lye Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Ser Val Ser WO 01/21647 PCTIBJO/01470 58 40 Ala Lys Gin Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro 55 Ile Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gin Gin 70 75 Val Leu Thr Ser Leu Pro Ser Gin Asn Val Leu Gin Ile Ala Asn Asp 90 Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser 100 105 110 Cys Ser Leu Pro Gin Thr Ser Gly Leu Gin Lys Pro Giu Ser Leu Asp 115 120 125 Gly Val Leu Giu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser 130 135 140 Arg Leu Gin Gly Ser Leu Gin Asp Ile Leu Gin Gin Leu Asp Ile Ser 145 150 155 160 Pro Giu Cys <210> 31 <211> 146 <212> PRT <213> Gorilla gorilla <400> 31 Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ile Val Thr Arg Ile Ser Asp Ile Ser His Thr Gin Ser Val Ser Ser 25 Lys Gin Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 40 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 55 Leu Thr Ser Met Pro Ser Arg Asn Met Ile Gin Ile Scr Asn Asp Leu 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 90 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala 5cr Giy Tyr Ser Thr Glu Val Val Ala Leu 5cr Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp met Leu Trp Gin Leu Asp Leu 5cr Pro 130 135 140 Gly Cys 145 <210> 32 <211> 167 <212> PRT <213> Homo sapiens <400> 32 Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu 1 5 10 Phe Tyr Val Gin Ala Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys 25 Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr 40 Gln Ser Val 5cr Ser Lys Gin Lys Val Thr Gly Leu Asp Phe Ile Pro 55 Gly Leu His Pro Ilie Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala 70 75 Val Tyr Gin Gin Ile Leu Thr Scr Met Pro Ser Arg Asn Val Ile Gin 90 Ile Ser Asn Asp Leu Giu Asn Leu Arg Asp Leu Leu His Val Leu Ala WO 01/21647 WOOI/1647PCTJIBOO/0 1470 105 110 Leu Pro Trp Ala Ser Gly Leu Giu Thr Leu 120 125 Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val 135 140 Gin Gly Ser Leu Gin Asp Met Leu Trp Gin 155 160 Cys <210> 33 <211> 167 <212> PRT <213> Macaca mulatta <400> 33 Met Tyr Trp Arg Thr I 1 Phe Tyr Ile Gin Ala 1 Thr Leu Ile Lys Thr 3 Gin Ser Val Ser Ser I dly Leu His Pro Val I Ile Tyr Gin Gin Ile I Ile Ser Asn Asp Leu.C 100 Phe Ser Lys Ser Cys I- 115 Giu Ser Leu Gly Asp 1 130 Val Ala Leu Ser Arg 1 145 3 Leu Asp Leu Ser Pro C 165 <210> 34 <211> 167 <212> PRT <213> Mus musculus <400> 34 Met Cys I) 1 Ser Tyr I Thr LeuI Gin Ser N Gly Leu 1 Val Tyr C Ie Ala Phe Ser I

I

Giu Ser 1 130 WO 01/21647 PCT/tB00/01 470 Val Ala Leu Ser Arg Leu Gin Gly Ser Leu Gin Asp Ile Leu Gin Gin 145 150 155 160 Leu Asp Val Ser Pro Glu Cys 165 <210> <211> 146 <212> PRT <213> Ovus aries <400> Val Pro Ilie Arg Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ile Val. Thr Arg Ilie Asn Asp Ilie Ser His Thr Gin Ser Val Ser Ser 25 Lys Gin Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Leu 40 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala Ile Tyr Gin Gin Ile 55 Leu Ala Ser Leu Pro Ser Arg Asn Vai Ile Gin Ile Ser Asn Asp Leu 70 75 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser Cys 90 Pro Leu Pro Gin Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val 100 105 110 Val Leu Giu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Arg Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 <210> 36 <211> 146 <212> PRT <213> Pan troglodytes <400> 36 Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ilie Val Thr Arg Ilie Asn Asp Ile Ser His Thr Gin Ser Val Ser Ser 25 Lys Gin Lys Val Thr Giy Leu Asp Phe Ile Pro Gly Leu His Pro Ile 40 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 55 Leu Thr Ser Met Pro Ser Arg Asn Met Ile Gin Ile Ser Asn Asp Leu 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 90 His Leu Pro Trp, Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Giu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 <210> 37 <211> 146 <212> PRT <213> Pongo pygmaeus WO 01/21647 WO 0121647PCT/IBOO/01470 <400> 37 Val Pro Ie Gin Lys Val 1 Val Ile Thr Arg Ile Asn Lys Gin Lys Val Thr Gly Leu Thr Leu Ser Lys Met Leu Thr Ser Met Pro Ser 70 Glu Asn Leu Arg Asp Leu His Leu Pro Trp Ala Ser 100 Vai Leu Giu Ala Ser Gly 115 Leu Gin Arg Ser Leu Gin 130 Gly Cys 145 <210> 38 <211> 167 <212> PRT <213> Rattus norvegicus Thr Ser Ile Ile Leu Cys Gly Arg Pro Leu Lys Thr Pro Ala Gin Ala Pro Val1 Gin 160 <400> 38 Leu Val Ile Arg Leu Leu Giu Ser Val Leu 150 Giu <210> 39 <211> 167 <212> PRT <213> Sus scrofa <400> 39 Met Arg Cys Gly Pro 1 5 Ser Tyr Val Glu Ala Thr Leu Ilie Lys Thr Cys Arg Phe Leu Trp Leu Trp Pro Tyr Leu 10 Pro Ile Trp Arg Vai Gin Asp Asp Thr Lys 25 Val Thr Arg Ile Ser Asp Ile Ser His Met WO 01/21647 WO 0121647PCT/IBOO/01 470 Val Thr Ser Lys Leu Pro Arg Asp 105 Gin Ala Ala Ser Ala Leu <210> <211> 4 <212> PRT <213> Homo sapiens <400> Glu Thr Leu Asp 1 <210> 41 <211> 4 <212> PRT <213> Mus musculus <400> 41 Gin Lys Pro Glu 1 <210> 42 <211> 6 <212> PRT <213> H-omo sapiens <400> 42 Leu Asp Ser Leu Gly Gly 1 <210> 43 <211> 4 <212> PRT <213> Homo sapiens <400> 43 Glu Lys Leu Glu 1 <210> 44 <211> 4 <212> PRT <213> Homo sapiens <400> 44 Glu Lys Pro Glu 1 WOO01/21647 PCT/IBOO/01470 63 <210> <211> 4 <212> PRT <213> Homo sapiens <400> Glu Lys Pro Asp 1 <210> 46 <211> <212> PRT <213> Homo sapiens <400> 46 Thr Pro Asp Ser Leu 1 <210> 47 <211> 9 <212> PRT <213> Homo sapiens <400> 4? Gly Leu Gin Thr Leu Asp Ser Leu Gly 1 <210> 48 <211> <212> PRT <213> Homo sapiens <400> 48 Gly Gly Val Leu Glu 1 <210> 49 <211> 6 <212> PRT <213> Homo sapiens <400> 49 Thr Pro Asp Ser Leu Gly 1 <210> <211> 9 <212> PRT <213> Homo sapiens <400> Ser Leu Gly Gly Val Leu Glu Ala Ser 1 <210> 51 <211> 6 <212> PRT <213> Homo sapiens <400> 51 Pro Glu Ser Leu Gly Gly 1 WO 01/21647 PCT/IBOO/01470 64 <210> 52 <211> 6 <212> PRT <213> Homo sapiens <400> 52 Pro Asp Ser Leu Gly Gly 1 <210> 53 <211> 7 <212> PRT <213> Homo sapiens <400> 53 Leu Gly Gly Val Leu Glu Ala 1 <210> 54 <211> 22 <212> PRT <213> Homo sapiens <400> 54 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 1 5 10 His Leu Pro Trp Ala Ser <210> <211> 22 <212> PRT <213> Homo sapiens <400> Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala 1 5 10 Ser Gly Leu Glii Thr Leu <210> 56 <211> 22 <212> PRT <213> Homo sapiens <400> 56 Ala Phe Ser Lys Ser Cys His Leu Pro Trp, Ala Ser Gly Leu Glu Thr 1 5 10 Leu Asp Ser Leu Gly Gly <210> 57 <211> 22 <212> PRT <213> Homo sapiens <400> 57 Cys His Leu Pro Trp Ala Ser Gly Leu Glii Thr Leu Asp Ser Leu Gly 1 5 10 Gly Val Leu Glu Ala Ser <210> 58 WO 01/21647 PCT/IBOO/01470 <211> 18 <212> PRT <213> Homo sapiens <400> 58 Leu Pro Trp, Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val 1 5 10 Leu Glu <210> 59 <211> 14 <212> PRT <213> Homo sapiens <400> 59 Trp Ala Ser Gly Leu 1 <210> <211> 21 <212> PRT <213> Homo sapiens <400> Ala Ser Gly Leu Glu 1 5 Gly Tyr Ser Thr Glu <210> 61 <211> <212> PRT <213> Homo sapiens <400> 61 Ser Gly Leu Glu Thr 1 5 <210> 62 <211> 22 <212> PRT <213> Homo sapiens Glu Thr Leu Asp Ser Leu Gly Gly Val Thr Asp Ser Leu Gly Gly Val Leu Glu Ala Ser 10 Leu Asp Ser Leu Gly <400> 62 Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr 1 5 10 Glu Val Val Ala Leu Ser <210> 63 <211> 22 <212> PRT <213> Homo sapiens <400> 63 Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 1 5 10 is Ser Arg Gly Gin Gly Ser <210> 64 <211> 22 <212> PRT WO 01/21647 PCT/IBOO/01470 66 <213> Mus musculus <400> 64 Giu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys 1 5 10 Ser Leu Pro Gin Thr Ser <210> <211> 22 <212> PRT <213> Mus musculus <400> Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys Ser Leu Pro Gin Thr 1 5 10 Ser Gly Leu Gin Lys Pro <210> 66 <211> 22 <212> PRT <213> Mus musculus <400> 66 Ala Phe Ser Lys Ser Cys Ser Leu Pro Gin Thr Ser Gly Leu Gin Lys 1 5 10 Pro Glu Ser Leu Asp Gly <210> 67 <211> 22 <212> PRT <213> Mus musculus <400> 67 Cys Ser Leu Pro Gin Thr Ser Gly Leu Gin Lys Pro Glu Ser Leu Asp 1 5 10 Gly Val Leu Glu Ala Ser <210> 68 <211> 18 <212> PRT <213> Mus musculus <400> 68 LeU Pro Gin Thr Ser Gly Leu Gin Lys Pro Glu Ser Leu Asp Gly Val 1 5 10 Leu Glu <210> 69 <211> 14 <212> PRT <213> Mus musculus <400> 69 Gin Thr Ser Gly Leu Gin Lys Pro Glu Ser Leu Asp Gly Val 1 5 <210> <211> 22 <212> PRT WO 01/21647 PCT/IBOO/0 1470 67 <213> Mus musculus <400> Thr Ser Gly Leu Gin Lys Pro Glu Ser Leu Asp Gly Val Leu Glu Ala 1 5 10 Ser Leu Tyr Ser Thr Glu <210> 71 <211> <212> PRT <213> Mus musculus <400> 71 Ser Gly Leu Gin Lys Pro Glu Ser Leu Asp 1 5 <210> 72 <211> 22 <212> PRT <213> Mus musculus <400> 72 Lys Pro Glu Ser Leu Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr 1 5 10 Glu Val Val Ala Leu Ser <210> 73 <211> 22 <212> PRT <213> Mus musculus <400> 73 Asp Gly Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu 1 5 10 Ser Arg Leu Gin Gly Ser <210> 74 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Chimeric oligonucleotides <400> 74 atgcaacagg acggacttgg agtagttttc uacuccaagt cagtccuguu gcaugcgcgt ttcgcgc 67 <210> <211> <212 DMNA <213> Artificial Sequence <220> <223> oligonucleotide Forward Primer <400> tgtccacgtc gtttacgctc <210> 76 WO 01/21647 PCTJIBOO/01470 68 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Reverse Primer <400> 76 tcccacttcc gttccttgtc <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Probes endogenouB/mutant <400> 77 cctactccaa gtcmgtcctg ttgcatt 27 <210> 78 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Chimeric oligonucleotides <400> 78 gaccctgccc tgtacctacc taccagatgt tttcaucugg uaggttcagg gcagggucgc gcgtttt 67 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Forward Primer <400> 79 gtggtgatcc tcttccagcc t 21 <210> <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Reverse Primer <400> ct~cj te2Sttgc 19 <210> 81 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Probes endogenous/mutant WO 01/21647 PCTIIBOO/01470 69 <400> 81 accctgccct gwcctaccag atgac <210> 82 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Chimeric oligonucleotides <400> 82 tggctgagct cttacctggt tttcattttt gaaaaccagg tcagagctca gccagcgcgt tttcgcgc 68 <210> 83 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Forward Primer <400> 83 gagctcatcg tccttgggag <210> 84 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Reverse Primer <400> 84 agtcttctat gggccccgc 19 <210> <211> 27 <212> DNA <213> Artificial sequence <220> <223> oligonucleotide Probes endogenous /mutant <400> caccgactcg agamtggacc aaaagtc 27 <210> 86 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Chimeric oligonucleotides <400> 86 ggttgtggta tgcctggctg ccttcttttg aaggcagcca gtcataccac aaccgcgcgt tttcgcgc 68 <210> 87 <211> <212> DNA WO 01/21647 <213> Artificial Sequence <220> <223> oligonucleotide Forward Primer <400> 87 acgcagagct catcgtcctt <210> 88 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Reverse Primer <400> 88 gatgcccagg aggaggaaga <210> 89 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Probes endogenous/mutant <400> 89 caacaccata ckgaccgacg gaa <210> <211> 18 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide mouse LSR specific primer <400> acgcatggga atcatggc <210> 91 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Zinc finger nuclotides of SEQID1 <400> 91 taggggtgag cggcgggg <210> 92 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Zinc finger nuclotides of SEQID1 <220> <221> misc feature <222> 10..12 PCT/IB00/01470 23 18 18 WOO01/21647 71 <223> n=a, g, c or t <400> 92 gagggctggn nntaggggtg a <210> 93 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Zinc finger nuclotides of SEQIDI <220> <221> misc feature <222> 10. .11 <223> n-a, g, c or t <400> 93 agggctgggn ntaggggtga <210> 94 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Zinc finger nuclotides of SEQIDi <400> 94 gtgggagccg agggctgg <210> <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide zinc finger nuclotides of SEQIDI <220> <221> misc feature <222> <223> n=a, g, c or t <400> gtgggagccn agggctggg <210> 96 <211> 1B <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Zinc finger nuclotides of SEQIDi <400> 96 gcggcggccg ggtgggag <210> 97 <211> 18 <212> DNA <213> Artificial Sequence PCTIBOO/O 1470 21 18 19 18 WO 01/21647 PCT/IBOO/01470 72 <220> <223> oligonucleotide Zinc finger nuclotides of SEQIDI <400> 97 ttggccggag cagatggg 18 <210> 98 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide zinc finger nuclotides of SEQID1 <220> <221> misc feature <222> 10. .11 <223> n=a, g, c or t <400> 98 gcagatgggn nccggaaggg <210> 99 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Zinc finger nuclotides of SEQIDI <220> <221> misc feature <222> 10. .12 <223> n~a, g, c or t <400> 99 agggctgggn nnaggggtga g 21 <210> 100 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide Zinc finger nuclotides of SEQIDi <220> <221> misc feature <222> 10. .12 <223> n=a, g, c or t <400> 100 Aggggitcaan rncagggagg a 21 <210> 101 <211> 18 <212> DNA <213> Artificial Sequence 4220> <223> oligonucleotide Zinc finger nuclotides of SEQIDi WO 01/21647 73 <400> 101 aagtgggtct cggttgca <210> 102 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide zinc finger LSR sequences <400> 102 aaggtcgcct atggtgcaga c <210> 103 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide zinc finger LSR sequences <400> 103 gtgggagccc gggggctgga <210> 104 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide zinc finger LSR sequences <400> 104 tgggggtggg cggcgggg <210> 105 <211> <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide zinc finger LSR sequences <400> 105 ccgggagtgc gcagggggta <210> 106 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide zinc finger LSR sequences <400> 106 gtggctgcac aaggtcgcc PCT/IB00/01470 18 21 18

Claims (22)

1. A leptin polypeptide fragment that modulates an activity of LSR, comprising at least 4, but not more than 50 contiguous amino acids of a polypeptide sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, wherein said at least 4 and not more than 50 contiguous amino acids include the leptin fragment central sequence.

2. The fragment of claim 1, comprising at least 10, but not more than 50 contiguous amino acids of said polypeptide sequence, wherein said at least 10 and not more than 50 contiguous amino acids include said leptin fragment central sequence. 15 3. The fragment of claim 2, comprising at least 20, but not more than 40 contiguous amino acids of said polypeptide sequence, wherein said at least 20 and not more than 40 contiguous amino acids include said leptin fragment central sequence.

4. The fragment of claim 3, comprising at least 20, but not more than 30 contiguous 20 amino acids of said polypeptide sequence, wherein said at least 20 and not more than 30 contiguous amino acids include said leptin fragment central sequence. The fragment of claim 1, comprising a 22 contiguous amino acid sequence, that is at least 75% identical to said leptin fragment variable region of said 25 polypeptide sequence.

6. The fragment of claim 5, wherein said 22 contiguous amino acid sequence is at least 85% identical to said leptin fragment variable region.

7. The fragment of claim 6, wherein said 22 contiguous amino acid sequence is at least 95% identical to said leptin fragment variable region of said polypeptide sequence.

8. The fragment of any one of claims 1 to 7, wherein said polypeptide sequence is SEQ ID NO:32. 113

9. The fragment of any one of claims 1 to 7, wherein said polypeptide sequence is SEQ ID NO:34. A polynucleotide encoding said leptin fragment of any one of claims 1 to 9, or the complement of a polynucleotide encoding said leptin fragment of any one of claims 1 to 9.

11. A recombinant cell comprising said polynucleotide of claim

12. A recombinant vector comprising said polynucleotide of claim 11.

13. A recombinant cell comprising said vector of claim 12.

14. A pharmaceutical composition comprising said leptin fragment of any one of 15 claims 1 to 9 and a pharmaceutically acceptable diluent.

15. A method of preventing or treating an obesity-related disease or disorder S-comprising providing to an individual in need of such treatment said pharmaceutical composition of claim 14.

16. The method of claim 15, wherein said obesity-related disease or disorder is selected from the group consisting of obesity, anorexia, cachexia, cardiac insufficiency, coronary insufficiency, stroke, hypertension, atheromatous disease, atherosclerosis, high blood pressure, non-insulin-dependent diabetes, 25 hyperlipidemia, hyperuricemia, congenital generalized lipodystrophy, and Syndrome X.

17. The method of claim 15 or claim 16, wherein said individual is a mammal.

18. A method of designing mimetics of a leptin fragment that modulates an activity of LSR, comprising: identifying critical interactions between one or more amino acids of said leptin fragment of any one of claims 1 to 9 and LSR; designing potential mimetics to comprise said critical interactions; and testing said potential mimetics ability to modulate said activity as a means for designing said mimetics. 114

19. The method of claim 18, wherein said activity is selected from the group consisting of leptin binding, leptin uptake, leptin degradation, triglyceride binding, triglyceride uptake, and triglyceride degradation.

20. The method of claim 18 or claim 19, wherein said critical interactions are selected from the group consisting of hydrogen bonding, covalent bonding, Van der Waals forces, steric hindrances, and hydrophobic interactions.

21. The method according to any one of claims 18 to 20, wherein said critical interactions are identified using assays selected from the group consisting of NMR, X-ray crystallography, and computer modeling.

22. Use of a leptin polypeptide fragment according to any one of claims 1 to 9 for the manufacture of a medicament for preventing or treating an obesity-related disease or disorder in an individual.

23. The use of claim 22, wherein said obesity-related disease or disorder is selected from the group consisting of obesity, anorexia, cachexia, cardiac insufficiency, coronary insufficiency, stroke, hypertension, atheromatous disease, 20 atherosclerosis, high blood pressure, non-insulin-dependent diabetes, hyperlipidemia, hyperuricemia, congenital generalized lipodystrophy, and Syndrome X.

24. The use of claim 22 or claim 23, wherein said individual is a mammal.

25. A leptin polypeptide fragment that modulates an activity of LSR according to claim 1, substantially as hereinbefore described with reference to the Examples, excluding, if any, comparative Examples. Dated this twenty-third day of May 2005 Serono Genetics Institute S.A. Patent Attorneys for the Applicant: F B RICE CO